Construction method and application of exogenous gene cluster chromosome integration system in streptomyces

Abstract

The invention belongs to the technical field of biology, and particularly relates to a construction method and application of an exogenous gene cluster chromosome integration system in streptomyces. The invention reports a new way of integrating an exogenous gene cluster onto a streptomyces chromosome, namely RMCE. The RMCE reduces the influence of other DNA sequences on the stable existence of the gene cluster as much as possible; secondly, the RMCE landing site still has functions after recombination and can be reused; in addition, lox71 and lox66 sites are added on a Dre, Vika and PhiBT1 mediated integration vector for knockout of the resistance gene of the apolamycin, so that the resistance gene can be conveniently used for multiple times. In addition, the recombinase Cre, the recombinase Dre and the recombinase Vika are used for integrating gene clusters in the streptomycete for the first time, the function of the tyrosine recombinase in the streptomycete is expanded, and therefore the tyrosine recombinase has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for constructing an exogenous gene cluster chromosomal integration system in Streptomyces and its application, mainly relating to a method for constructing an RMCE system mediated by Vika, Dre, Cre, and PhiBT1 and its application. Background Art

[0002] Studies on the biosynthesis of natural products have shown that genes responsible for biosynthesis, self-resistance, regulation, and transport are usually physically clustered in the genome to form biosynthetic gene clusters (BGCs). Among actinomycetes, the strain Streptomyces coelicolor A3(2) genomic information (GenBank: AL645882) was completely sequenced and annotated in 2002, and its main metabolites include red prodiginine encoded by the gene cluster, act actinorhodin, an aromatic polyketide encoded by the gene cluster, and cda calcium-dependent antibiotic, a non-ribosomal peptide encoded by the gene cluster, etc. Due to the rich precursors, extensive synthetic pathways, and available genetic engineering, S. coelicolor A3(2) and its derivatives have been used as hosts for heterologous expression of natural product synthetic gene clusters. Various types of natural products such as polyether antibiotic salinomycin, lantibiotic erythreapeptin, and meroterpenoid antibiotic merochlorins have been successfully expressed in S. coelicolor A3(2) or its derivatives.

[0003] Natural product synthetic gene clusters are generally integrated into the Streptomyces chromosome by serine recombinase-mediated site-specific recombination for heterologous expression. On the S. coelicolor chromosome of A3(2), there is a attB site that can be recognized by the recombinase PhiC31 and is often used as the insertion site for exogenous gene clusters. Under the action of the recombinase, the attP site on the gene cluster-carrying vector and the attBRecombination occurs at the locus, integrating all DNA sequences on the entire plasmid into the chromosome. To reduce the integration of unnecessary DNA sequences on the plasmid, a new integration method needs to be developed in Streptomyces, such as the recombinase-mediated cassette exchange (RMCE) technology to achieve site-specific integration of gene clusters. RMCE utilizes site-specific recombinases and a pair of sites that do not recombine with each other and can be recognized by the same recombinase to achieve the purpose of DNA cassette exchange. Indra Roux and Yit-Heng Chooi developed an RMCE technology based on Cre / lox for the system of targeted integration of foreign gene clusters into chromosomes. First, an RMCE landing site loxP and lox2272 were constructed on the chromosome. Aspergillus nidulans , lox2272 is a base mutation in the spacer sequence of loxP . Different loxP and lox2272 at the spacer region do not recombine under the action of Cre enzyme; then, loxP and lox2272 sites were placed at both ends of the target gene cluster; the vector containing the gene cluster was transferred into Aspergillus nidulans containing the RMCE landing site. ， Under the action of the recombinase Cre, loxP-lox2272 on the vector recombines with loxP-lox2272 on the chromosome to complete the insertion of the gene cluster. This is the first application of RMCE technology in fungi to participate in the integration of gene clusters. In addition to loxP and lox2272 mutation sites, rox and the mutation site rox2232, vox and the mutation site vox2261 also do not recombine under the action of the recombinases Dre or Vika. Therefore, it is expected to develop a Cre, Dre or Vika-mediated RMCE system for the integration of gene clusters in Streptomyces. However, the inventors found that there have been no reports in the relevant literature on the integration of gene clusters using Cre, Dre or Vika-mediated RMCE systems in Streptomyces. Summary of the Invention

[0004] Aiming at the deficiencies of the above-mentioned existing technologies, the purpose of the present invention is to provide a method for constructing an exogenous gene cluster chromosomal integration system in Streptomyces and its application. Specifically, the present invention constructs an RMCE system that can be used in Streptomyces, mainly including two aspects: constructing a strain of Streptomyces containing 4 RMCE landing sites (vox-vox2261, rox-rox2232, lox5171-lox2272, and attB-attB15) S. coelicolor A3(2)-2023 and constructing 4 integration vectors containing Vika, Dre, Cre, and PhiBT1 respectively for the integration of exogenous gene clusters. Based on the above research results, the present invention is completed.

[0005] Specifically, the technical solution of the present invention is as follows: In the first aspect of the present invention, a method for constructing an exogenous gene cluster chromosomal integration system in Streptomyces is provided. The construction method includes: constructing Streptomyces containing RMCE landing sites; and constructing integration vectors containing Vika, Dre, Cre, and PhiBT1 respectively for the integration of exogenous gene clusters; wherein, the RMCE landing sites include vox-vox2261, rox-rox2232, lox5171-lox2272, and attB-attB15.

[0006] Among them, the RMCE landing sites vox-vox2261, rox-rox2232, lox5171-lox2272, and attB-attB15 are respectively inserted into the endogenous gene clusters of Streptomyces red, act, cda and clb positions.

[0007] More specifically, four pairs of different recombinase target sites (RTS), namely vox-vox2261, rox-rox2232, lox5171-lox2272, and attB-attB15, are used as the RMCE landing sites and are successively inserted into 4 endogenous gene clusters red , act , cda and clb positions. The DNA sequences of the four pairs of sites are shown in Table 1. The DNA sequences of the endogenous gene clusters are disrupted, which not only simplifies the metabolic background of Streptomyces itself but also reduces the metabolic competition with exogenous genes, enabling more precursors and energy to flow to the target gene cluster; the insertion of the RMCE landing sites provides a new integration method for the integration of exogenous gene clusters, and the insertion of multiple landing sites provides sites for the multi-copy integration of exogenous gene clusters, which is beneficial to the improvement of the yield of natural products and the discovery of new compounds.

[0008] Table 1 DNA sequences of four pairs of RMCE landing sites

[0009] Note: The bold letters are the spacer regions of the sites.

[0010] Specifically, the method for constructing Streptomyces containing RMCE landing sites may include: using Streptomyces S. coelicolor A3(2) as the starting strain to construct S. coelicolor A3(2)-2023, More specifically, the construction method includes: 1) Constructed a plasmid for RMCE landing site insertion and gene cluster knockout ( Figure 2 ); 2) Transfer the plasmid into S. coelicolor Strain A3(2) by conjugation transfer, culture at 39 °C or in a medium without thiostrepton to promote the occurrence of double crossover. The apramycin (apra) resistance DNA fragment RTS1-lox71-apra-lox66-RTS2 with RMCE landing sites (RTS1 and RTS2) is integrated into the chromosome, and the corresponding gene cluster is knocked out; 3) Transfer the plasmid pUWLCRE containing Cre recombinase into the strain with the gene cluster knocked out. Under the action of Cre, lox71 and lox66 on the chromosome recombine, and the apramycin (apra) resistance gene is deleted, leaving only the active RMCE landing site RTS1-RTS2.

[0011] 4) Insert vox-vox2261, rox-rox2232, lox5171-lox2272, and attB-attB15 into red , act , cda and clb gene cluster positions in sequence in the above manner to construct S. coelicolor A3(2)-2023. The S. coelicolor A3(2)-2023 finally deleted a total of 159 kb and inserted the above 4 pairs of RMCE landing sites.

[0012] Among them, in step 1), the plasmid contains 4 parts: HaF is a fragment containing a 2-kb homologous arm upstream of the target region, HaR is a fragment containing a 2-kb homologous arm downstream of the target region, RTS1-apra-RTS2 is a fragment containing the apramycin (apra) resistance gene with RMCE landing sites, and a replicon fragment containing the transfer origin oriT and the thiostrepton (tsr) resistance gene.

[0013] Construct integration vectors containing Vika, Dre, Cre, and PhiBT1 respectively for the integration of exogenous gene clusters. Specifically, taking the xiamenmycin gene cluster xim as an example of the exogenous gene cluster, the specific integration method includes ( Figure 3 ): 1) Construct an integration vector of the xiamenmycin gene cluster containing different recombinases; assemble 5 fragments, pSG5-amp-tsr, BAC, int-RTS1, xim, and apra-RTS2, by the ExoCET technique ( Figure 4 ); each vector contains: the xiamenmycin gene cluster xim (which is derived from Streptomyces xiamenensis S. xiamenensis 318); int, one of the four recombinases vika, dre, cre, and phiBT1, two non-recombinogenic sites RTS1 and RTS2 recognized by the recombinase; the apramycin resistance gene apra, used for screening after recombination in Streptomyces; the thiostrepton resistance gene tsr, used to verify plasmid elimination; psG5 is a temperature-sensitive replicon and the origin of transfer oriT.

[0014] 2) Transfer the integration vector into S. coelicolor A3(2)-2023 by conjugation transfer, and culture it at 39 °C. The recombinase functions to mediate the recombination of RTS1-RTS2 on the plasmid with RTS1-RTS2 on the chromosome, completing the site-directed insertion of the xiamen gene cluster. At this time, the strain is named S. coelicolor A3(2)-2023-xim-apra.

[0015] 3) Transfer the plasmid pUWLCRE containing the Cre recombinase into S. coelicolor A3(2)-2023-xim-apra, lox71 and lox66 recombination occurs, and the apramycin resistance gene is knocked out, facilitating the next use of this resistance gene. Construct a strain containing the xiamenmycin gene cluster without the presence of resistance markers. At this time, the strain is named S. coelicolor A3(2)-2023-xim.

[0016] Further, in step 1), four integrative vectors of the xiamenmycin gene cluster containing different recombinases (Vika-integrative vector, Dre-integrative vector, Cre-integrative vector, and PhiBT1-integrative vector) are constructed. Their characteristics are shown in Table 2. The nucleotide sequences of the four recombinases Vika, Dre, Cre, and PhiBT1 on the vector are shown in SEQ ID NO.9-12 respectively. Under the same genetic background, the target sites of different recombinases are specific and no cross-recombination will occur. Therefore, except for Cre, other recombinases cannot mediate lox recombination between lox71 and lox66 sites. Therefore, there are

[0017] Table 2 Four integrative vectors of the xiamenmycin gene cluster

[0018] Further, by using the four integrative vectors constructed by the above method, the integration of one to four copies of the xiamenmycin gene cluster on the S. coelicolor chromosome of A3(2)-2023 can be achieved. By comparing the xiamenmycin yields, it is found that the purpose of increasing the yield can be achieved by increasing the gene cluster copy number.

[0019] In the second aspect of the present invention, a Streptomyces genetic engineering bacterium obtained by the above construction method is provided. The Streptomyces genetic engineering bacterium integrates one or more copies of foreign genes or foreign gene clusters; the multiple can be 2, 3, or 4.

[0020] Therefore, in the third aspect of the present invention, the application of the above construction method or Streptomyces genetic engineering bacterium in any one or more of the following is provided: (a) Functional research of foreign genes or foreign gene clusters; (b) Improvement of natural product yields; (c) Discovery of new compounds.

[0021] The beneficial technical effects of the above one or more technical solutions: The above technical solution discloses a new method for integrating exogenous gene clusters into Streptomyces chromosomes - RMCE. This is the first time that RMCE technology has been used in Streptomyces to participate in the integration of gene clusters, and the chromosome integration of 4 copies of the xiamenmycin gene cluster has been completed. Compared with the traditional site-specific recombination mediated by PhiC31 to integrate all DNA sequences on the plasmid containing the gene cluster into the chromosome, RMCE integrates the exogenous gene cluster into the chromosome in the form of a cassette, and only integrates the required DNA sequence, avoiding the integration of other DNA sequences on the plasmid including replicons, integrases and transfer elements oriT, and minimizing the impact of other DNA sequences on the stable existence of the gene cluster; secondly, the RMCE landing site is consistent with the PhiC31 recognition site. attB The difference between the two sites is that the RMCE landing site remains functional after recombination and can be reused, while attB After recombination, it loses its function; and in the integration vectors mediated by Dre, Vika and PhiBT1, lox71 and lox66 The site is used to knock out the apramycin resistance gene, which facilitates the multiple use of a resistance gene. In addition, this is the first time that the recombinases Cre, Dre and Vika have been used for the integration of gene clusters in Streptomyces, expanding the function of tyrosine recombinase in Streptomyces. RMCE-mediated multi-copy chromosome integration expands the toolbox of synthetic biology in Streptomyces, and therefore has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0023] Figure 1 : S. coelicolor Schematic diagram of the construction process of A3(2)-2023.

[0024] Figure 2 : Schematic diagram of the construction of plasmids for RMCE landing site insertion and gene cluster knockout.

[0025] Figure 3 :Xiamenmycin is integrated into S. coelicolor Schematic diagram of A3(2)-2023 on chromosome.

[0026] Figure 4 :Schematic diagram of the construction process of RMCE integration vector.

[0027] Figure 5 : Expression levels of strains containing different copies of the xiamenmycin gene cluster. DETAILED DESCRIPTION

[0028] It should be noted that the following detailed description is illustrative and aims to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

[0029] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0030] The present invention will be further described in conjunction with specific examples. All strains and plasmids involved in the following examples, unless otherwise specified, are derived from the research group of Zhang Youming at Shandong University. The DNA sequences of the recombinases Vika, Dre, Cre, and PhiBT1 in the RMCE vector involved in the examples are shown in SEQ ID NO. 9-12. The experimental methods and reagents in the examples, unless otherwise specified, are conventional methods and commercially available reagents in the art and can be obtained through commercial channels.

[0031] Example 1: Construction of plasmids for insertion at the vox-vox2261 locus and red gene cluster knockout For insertion at the vox-vox2261 locus and red The gene cluster knockout plasmid pKC1139-red-vox-apra-vox2261 was assembled from 4 target fragments HaF, HaR, vox-apra-vox2261, and pKC1139-tsr each with a 40 bp homologous arm. After preparing the 4 DNA fragments according to Table 3, they were purified using an agarose gel recovery kit, and the purified fragments were subjected to ExoCET assembly ( Nucleic Acids Res . 2018, 46(5): e28), and screened using LB medium containing apramycin. Table 4 shows the primer sequences required for preparing the DNA fragments. The construction methods of the other 3 knockout plasmids are the same as that of pKC1139-red-vox-apra-vox2261. Table 5 shows the preparation of the DNA fragments required for the plasmid pKC1139-act-rox-apra-rox2232 with the rox-rox2232 locus for act gene cluster knockout; Table 6 shows the preparation of the DNA fragments required for the plasmid with the lox5171-lox2272 locus for cdaPreparation of DNA fragments required for plasmid pYH7-cda-lox5171-apra-lox2272 for gene cluster knockout; Table 7 shows the plasmid pYH7-clb-attB-apra-attB15 with attB-attB15 sites for clb preparation of DNA fragments required for gene cluster knockout.

[0032] Table 3 DNA fragments required for the construction of pKC1139-red-vox-apra-vox2261

[0033] Table 4 Primer sequences required for the construction of pKC1139-red-vox-apra-vox2261

[0034] Note: Lowercase letters are homologous arms.

[0035] Table 5 DNA fragments and primers required for the construction of pKC1139-act-rox-apra-rox2232

[0036] Note: Lowercase letters are homologous arms.

[0037] Table 6 DNA fragments and primers required for the construction of pYH7-cda- lox5171-apra-lox2272

[0038] Note: Lowercase letters are homologous arms.

[0039] Table 7 DNA fragments and primers required for the construction of pYH7-clb-attB-apra-attB15

[0040] Note: Lowercase letters are homologous arms.

[0041] Example 2: Construction of the integration vector Vika-integrative vector The integration vector Vika-integrative vector is assembled from 5 fragments: pSG5-amp-tsr, BAC, xim, Vika-vox, and apra-vox2261. After preparing 5 DNA fragments according to Table 8, they were purified using an agarose gel recovery kit, and the purified fragments were assembled by ExoCET ( Nucleic Acids Res. In 2018, 46(5): e28), screening was carried out using LB medium containing apramycin. Table 9 shows the primer sequences required for preparing DNA fragments. The construction methods of the other 3 integrative vectors are the same as those of the Vika-integrative vector. Table 10 shows the preparation of DNA fragments required for the integrative vector Dre-integrative vector; Table 11 shows the preparation of DNA fragments required for the integrative vector Cre-integrative vector; Table 12 shows the preparation of DNA fragments required for the integrative vector PhiBT1-integrative vector.

[0042] Table 8 DNA fragments required for the construction of the integrative vector Vika-integrative vector

[0043] Table 9 Primer sequences required for the construction of the integrative vector Vika-integrative vector

[0044] Note: Lowercase letters are homologous arms.

[0045] Table 10 DNA fragments and primers required for the construction of the integrative vector Dre-integrative vector

[0046] Note: Lowercase letters are homologous arms.

[0047] Table 11 DNA fragments and primers required for the construction of the integrative vector Cre-integrative vector

[0048] Note: Lowercase letters are homologous arms.

[0049] Table 12 DNA fragments and primers required for the construction of the integrative vector PhiBT1-integrative vector

[0050] Note: Lowercase letters are homologous arms.

[0051] According to the above method, using the 4 constructed integrative vectors, the Xiamenmycin gene cluster can be integrated into S. coelicolor 1 to 4 copies on the chromosome of A3(2)-2023 (Table 13).

[0052] Table 13 Genotypes of Strains with Xiamenmycin Gene Cluster Inserted

[0053] Example 3: Comparison of Xiamenmycin Yields Since there is no xiamenmycin available on the market currently and no standard product to prepare a standard curve, we integrated the gene cluster into the attB S. coelicolor site on the chromosome of A3(2)-2023 phiC31 ( S. coelicolor xim-attB phiC31 ) and used the peak area of xiamenmycin in the crude extract of the fermentation product as a control. The yields of xiamenmycin in different strains were compared by the peak area of the compound. It was found that the purpose of increasing the yield could be achieved by increasing the gene cluster copy number ( Figure 5 ). The specific experimental procedure for the relative quantitative analysis of xiamenmycin yield is as follows: 1) Streptomyces coelicolor containing the xim gene cluster on the antibiotic-free MS plate was transferred to a 250 mL Erlenmeyer flask containing 30 mL of TSB liquid medium and cultured at 30 °C and 200 rpm for 2 days as the seed solution.

[0054] 2) 1 mL of the seed solution was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of GYM liquid medium and cultured at 30 °C and 200 rpm for 6 days for fermentation.

[0055] 3) On the 6th day, 1 mL of macroporous resin XAD-16 suspension was added to the fermentation broth, and the culture was continued at 30 °C and 200 rpm for 12 hours.

[0056] 4) Centrifuge at 8500 rpm for 15 min, discard the supernatant, collect the cells and resin, add 30 mL of methanol (MeOH) to suspend, and pour it into a 250 mL Erlenmeyer flask. Incubate at 30 °C and 200 rpm for 6 h.

[0057] 5) Filter the MeOH extract through filter paper into a 250 mL round-bottom flask, and obtain the crude extract of the fermentation product by rotary evaporation.

[0058] 6) Dissolve the crude extract with 1 mL of MeOH, transfer it to a 1.5 mL centrifuge tube, centrifuge at 12000 rpm for 10 min, and filter the supernatant through a 0.22 μm organic phase filter membrane into an injection vial.

[0059] 7) HPLC detection and data acquisition, detection conditions: injection volume of 5 μL, flow rate of 0.6 mL / min, H2O with 0.1% (v / v) TFA as mobile phase A, acetonitrile (ACN) as mobile phase B. The elution program is as follows: 5% ACN from 0 to 3 min; 5 - 95% ACN from 3 to 18 min; 100% ACN from 18 to 22 min; 5% ACN from 22 to 25 min. The UV detection wavelength is 254 nm. The yield is represented by the peak area of xiamenmycin.

[0060] Matters not covered by this invention are well-known technologies.

[0061] The above embodiments are only for illustrating the technical concept and features of the present invention, and the purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and it should not be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for constructing a chromosome integration system of an exogenous gene cluster in Streptomyces, characterized in that: The construction method includes: constructing Streptomyces containing RMCE landing sites; and constructing integration vectors containing Vika, Dre, Cre and PhiBT1 respectively for the integration of exogenous gene clusters; wherein the RMCE landing sites include vox-vox2261, rox-rox2232, lox5171-lox2272 and attB-attB15.

2. The construction method according to claim 1, characterized in that: The RMCE landing sites vox-vox2261, rox-rox2232, lox5171-lox2272 and attB-attB15 were inserted into the endogenous gene cluster of Streptomyces, respectively. red, act, cda and clb Location.

3. The construction method according to claim 1, characterized in that: The method for constructing Streptomyces containing RMCE landing sites includes: using Streptomyces S. coelicolor A3(2) is the starting strain, and the RMCE landing site is constructed S. coelicolor A3(2)-2023.

4. The construction method according to claim 3, characterized in that: Methods for constructing Streptomyces containing RMCE landing sites include: 1) Plasmids for RMCE landing site insertion and gene cluster knockout were constructed; 2) Transfer the plasmid to S. coelicolor In strain A3(2), when cultured at 39°C or in a medium without thiostrepton, double crossover is promoted, and the apramycin-resistant DNA fragment RTS1-lox71-apra-lox66-RTS2 with the RMCE landing site (RTS1 and RTS2) is integrated into the chromosome, and the corresponding gene cluster is knocked out; 3) The plasmid pUWLCRE containing Cre recombinase is transferred into the strain with the knockout gene cluster. Under the action of Cre, the chromosome lox71 and lox66 Recombination occurs, and the apramycin resistance gene is deleted, leaving only the active RMCE landing site RTS1-RTS2; 4) Insert vox-vox2261, rox-rox2232, lox5171-lox2272 and attB-attB15 into red , act , cd and clb The location of the gene cluster is constructed to obtain S. coelicolor A3(2)-2023.

5. The construction method according to claim 4, characterized in that: In the step 1), the plasmid contains 4 parts: HaF is a fragment containing a homology arm 2 kb upstream of the target region, HaR is a fragment containing a homology arm 2 kb downstream of the target region, RTS1-apra-RTS2 is a fragment of the apramycin resistance gene with an RMCE landing site, and a replicon fragment containing the transfer initiation site oriT and the thiostrepton resistance gene.

6. The construction method according to claim 1, characterized in that: Construct integration vectors containing Vika, Dre, Cre and PhiBT1 respectively for the integration of exogenous gene clusters. Specifically, the specific integration method includes: 1) Construct an integration vector of the Xiamenmycin gene cluster containing different recombinases; assemble the five fragments pSG5-amp-tsr, BAC, int-RTS1, xim and apra-RTS2 using ExoCET technology; the integration vector contains: Xiamenmycin gene cluster xiim ; int, one of the four recombinases vika, dre, cre and phiBT1, two non-recombination sites RTS1 and RTS2 recognized by the recombinase; apramycin resistance gene apra, thiostrepton resistance gene tsr, temperature-sensitive replicon psG5 and conjugative transfer initiation site oriT. 2) Transfer the integration vector into S. coelicolor A3(2)-2023 was cultured at 39°C to mediate recombination between RTS1-RTS2 on the plasmid and RTS1-RTS2 on the chromosome, completing the site-specific insertion of the Xiamen gene cluster. At this time, the strain was named S. coelicolor A3(2)-2023-xim-apra; 3) Transfer the plasmid pUWLCRE containing Cre recombinase into S. coelicolor A3(2)-2023-xim-apra, lox71 and lox66 Recombination occurred, the apramycin resistance gene was knocked out, and a strain containing the xiamenmycin gene cluster without resistance markers was constructed. At this time, the strain was named S. coelicolor A3(2)-2023-xim.

7. The genetically engineered Streptomyces obtained by the construction method according to any one of claims 1 to 6.

8. The genetically engineered Streptomyces bacteria according to claim 7, characterized in that The genetically engineered Streptomyces bacteria are integrated with one or more copies of exogenous genes or exogenous gene clusters.

9. The genetically engineered Streptomyces bacteria according to claim 8, characterized in that The plurality is 2, 3 or 4.

10. Use of the construction method according to any one of claims 1 to 6 or the genetically engineered Streptomyces according to any one of claims 7 to 9 in any one or more of the following: (a) Functional studies of exogenous genes or exogenous gene clusters; (b) Improved yield of natural products; (c) Discovery of new compounds.

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