Application of an efflux gene sbi_04891-sbi_04892 in increasing the yield of macrolide antibiotics in Streptomyces

Abstract

An efflux gene sbi_04891‑sbi_04892 This invention relates to the application of increasing the yield of macrolide antibiotics in Streptomyces, falling under the field of genetic engineering technology. To improve the yield of macrolide antibiotics in Streptomyces, this invention discovered an efflux gene in *Streptomyces BC04* from Harbin. sbi_04891‑sbi_04892 The gene encodes an ABC transporter protein. By overexpressing this efflux gene in *Streptomyces hygroscopicus* BC04 and *Streptomyces bluegrayense* NMWT1, it was found that the efflux gene could increase the yield of milbemycin in *Streptomyces hygroscopicus* BC04 and the yield of nimodipine in *Streptomyces bluegrayense* NMWT1.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to an ectopic gene. sbi_04891-sbi_04892 Application in increasing the production of macrolide antibiotics by Streptomyces. Background Technology

[0002] Macrolide antibiotics are a class of biologically important secondary metabolites produced by Streptomyces. Many of them have been successfully developed into biopesticides and produced on a large scale industrially, with typical examples including milbemycin and nemadectin. However, the continuous increase in antibiotic fermentation yield per unit area leads to a large accumulation of the product within the bacterial cell. This accumulation effect severely restricts the normal growth and metabolism of the strain and limits further yield increases. Studies have shown that excessive accumulation of end products within the cell can have a negative feedback inhibitory effect on the strain's own biosynthetic pathway, thereby inhibiting the continued synthesis of antibiotics. In particular, when Streptomyces fermentation enters the stationary phase, the rate of antibiotic synthesis increases significantly, and the feedback inhibition caused by end product accumulation is particularly prominent at this time, becoming one of the key bottlenecks restricting the increase of antibiotic yield.

[0003] Efflux proteins are transport proteins located on the cell membrane that actively pump intracellular metabolites out of the cell, playing a crucial role in maintaining the dynamic balance of intracellular and extracellular substances. For antibiotic-producing bacteria, the pumping efficiency of efflux proteins directly affects the control of intracellular product concentration, thus influencing the utilization efficiency of precursor substances and the synthesis rate of the final product. Besides product secretion, enhancing the efflux of target products also helps reduce the difficulty of antibiotic isolation and extraction, simplifying downstream purification processes.

[0004] Current metabolic engineering modifications aimed at increasing antibiotic yield in Streptomyces mainly focus on optimizing biosynthetic pathways, modifying regulatory genes, and enhancing precursor supply, such as knocking out negative regulators or overexpressing positive regulators to increase antibiotic production. In contrast, the discovery of efflux genes and their application in constructing high-yielding antibiotic strains remains relatively limited. In particular, the systematic screening and identification of efflux genes targeting specific macrolide antibiotics in Streptomyces is insufficient. There is an urgent need to develop more efficient efflux gene resources to adapt and enhance antibiotic efflux pathways, providing new strategic support for further increasing the unit yield of industrial strains. Summary of the Invention

[0005] To increase the yield of macrolide antibiotics by Streptomyces, this invention discovered an efflux gene in Streptomyces BC04 from Harbin. sbi_04891-sbi_04892The gene encodes an ABC transporter protein. By overexpressing this efflux gene in *Streptomyces hygroscopicus* BC04 and *Streptomyces bluegrayense* NMWT1, it was found that the efflux gene could increase the yield of milbemycin in *Streptomyces hygroscopicus* BC04 and the yield of nimodipine in *Streptomyces bluegrayense* NMWT1.

[0006] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution: The first objective of this invention is to provide an efflux gene. sbi_04891-sbi_04892 Application in increasing the production of macrolide antibiotics by Streptomyces, the efflux gene sbi_04891-sbi_04892 The nucleotide sequence is shown in SEQ ID NO.1, and the macrolide antibiotic is milbemycin or nimocritin.

[0007] In one embodiment of the invention, the application is in *Streptomyces glabra* (…). Streptomyces bingchenggensis Overexpression in BC04 sbi_04891-sbi_04892 Genes, or in Streptomyces bluegrayense ( Streptomyces cyaneogriseus Overexpression in NMWT1 sbi_04891-sbi_04892 Gene.

[0008] The second objective of this invention is to provide a product containing the aforementioned efflux gene. sbi_04891-sbi_04892 Application of recombinant vectors in increasing the yield of macrolide antibiotics by Streptomyces.

[0009] In one embodiment of the present invention, the application involves introducing a recombinant vector into Escherichia coli and then transferring it into Streptomyces BC04 or Streptomyces NMWT1.

[0010] Further specifying, the *Escherichia coli* is... E.coli ET12567 / pUZ8002.

[0011] In one embodiment of the present invention, the method for constructing the recombinant vector includes the following steps: double digestion of plasmid pSET152 to obtain a linear vector backbone; using the genome of *Streptomyces BC04* as a template, amplification... sbi_ 04891-sbi_04892 Gene fragments and P hrdB Promoter fragment; linking the linear vector skeleton with sbi_04891-sbi_04892 Gene fragments and P hrdB The promoter fragments are spliced ​​together to obtain the recombinant vector; the P hrdB The nucleotide sequence of the promoter is shown in SEQ ID NO.2.

[0012] In one embodiment of the present invention, the amplification sbi_04891-sbi_04892The nucleotide sequences of the upstream and downstream primers used for the gene fragment are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively, for amplifying P. hrdB The nucleotide sequences of the upstream and downstream primers used for the promoter fragment are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0013] In one embodiment of the present invention, the restriction endonuclease used in the double digestion is: Xba I and Eco RI.

[0014] In one embodiment of the present invention, the ClonExpress MultiS kit is used to bond the linear vector backbone with... sbi_04891-sbi_04892 Gene fragments and P hrdB The promoter fragments are spliced ​​together.

[0015] A third object of the present invention is to provide a product containing the above-mentioned... sbi_04891-sbi_04892 Application of recombinant bacteria containing genes or the aforementioned recombinant vectors in increasing the production of macrolide antibiotics by Streptomyces.

[0016] In one embodiment of the present invention, the method for constructing the recombinant bacteria includes the following steps: constructing the above-mentioned... sbi_04891-sbi_04892 The gene overexpression integration plasmid was transformed into E. coli, and then the overexpression integration plasmid was introduced into the starting strain through intergeneric fusion transfer experiment to obtain recombinant bacteria.

[0017] In one embodiment of the present invention, the starting strain is *Streptomyces berberis* BC04 or *Streptomyces bluegrayense* NMWT1.

[0018] Further specifying, the *Escherichia coli* is... E.coli ET12567 / pUZ8002.

[0019] The beneficial effects of this invention are: This invention discloses the *Streptomyces hygroscopicus* (Bingcheng) Streptomyces bingchenggensis Efflux genes in BC04 sbi_04891 - sbi_04892 This gene encodes an ABC transporter protein, which can effectively promote the biosynthesis of macrolide antibiotics in Streptomyces. This invention uses *Streptomyces BC04* and *Streptomyces bluegray* NMWT1 as starting strains, respectively, and achieves this through overexpression... sbi_ 04891 - sbi_04892Genes were used to construct recombinant strains BC04 / Osbi_04891-04892 and NMWT1 / Osbi_04891-04892. It was found that when using recombinant strain BC04 / Osbi_04891-04892 for fermentation to produce milbemycin, compared to the control strain BC04 / pSET152, the milbemycin yield increased from 2531 mg / L to 3900.27 mg / L, a 0.5-fold increase. When using recombinant strain NMWT1 / Osbi_04891-04892 for fermentation to produce nimocritin, compared to the control strain NMWT1 / pSET152, the nimocritin yield increased from 310.12 mg / L to 672.31 mg / L, a 1-fold increase. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the construction of the recombinant plasmid pSET152::sbi_04891-04892; Figure 2 The graph shows the detection results of milbemycin content in the fermentation broth of recombinant strain BC04 / Osbi_04891-04892, control strain BC04 / pSET152, and original strain BC04. Figure 3 The graph shows the detection results of nimocriptine content in the fermentation broth of recombinant strain NMWT1 / Osbi_04891-04892, control strain NMWT1 / pSET152, and original strain NMWT1. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. In the art, any embodiments obtained by those skilled in the art without creative effort are protected by this invention.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The materials, reagents, culture media, and instruments used are all conventional materials, reagents, culture media, and instruments in the art, and can be obtained commercially by those skilled in the art. Unless otherwise specified, the molecular biology experimental operations involved in this invention, such as PCR amplification, enzyme digestion and ligation, and transformation, are all conventional experimental operations in the art or can be performed according to the product instructions of the corresponding reagents.

[0023] The plasmid information involved in the following examples is shown in Table 1; the strain information involved is shown in Table 2; and the primer information involved is shown in Table 3.

[0024] Table 1. Plasmid information involved in the examples

[0025] Table 2. Information on the strains involved in the examples.

[0026] Table 3 Primer information involved in the examples

[0027] The specific sources of *Streptomyces BC04* and *Streptomyces blue-gray* NMWT1 in Table 2 are as follows: Harbin Streptomyces ( Streptomyces bingchenggensis BC04 is the industrial-grade milbemycin production strain, disclosed in the following literature: ZHANG, Y. et al. Characterization of a pathway specific activator of milbemycin biosynthesis and improved milbemycin production by its overexpression in Streptomyces bingchenggensis [J]. Microb Cell Fact, 2016(15): 152.

[0028] blue-gray streptomyces ( Streptomyces cyaneogriseus NMWT1 is disclosed in the following literature: Li et al. China Life Sciences (2019) 15:152 DOI 10.1007 / s11427 018 9442 9.

[0029] The above-mentioned strains are available to the public from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

[0030] The culture medium involved in this invention is as follows: The solid culture medium required for the growth and fermentation of *Streptomyces hygroscopicus* in Harbin is SKYM medium, with the following composition: 4 g / L sucrose, 1 g / L skim milk powder, 2 g / L yeast extract, 5 g / L malt extract, 18 g / L agar powder, and the balance being water, pH 7.0-7.2.

[0031] The composition of the *Streptomyces hygroscopicus* seed culture medium in Harbin is as follows: 5 g / L yeast extract, 10 g / L sucrose, 1 g / L skim milk powder, 3.5 g / L bacterial peptone, 0.5 g / L dipotassium hydrogen phosphate, with the remainder being water, pH 7.2.

[0032] The composition of the fermentation medium for *Streptomyces hygroscopicus* in Harbin is as follows: 80 g / L sucrose, 20 g / L soybean meal, 1 g / L skim milk powder, 0.1 g / L ferrous sulfate, 1 g / L dipotassium hydrogen phosphate, 3 g / L calcium carbonate, with the remainder being water, pH 7.2.

[0033] The solid culture medium required for the growth and fermentation of *Streptomyces blue-gray* is ISP3 medium, with the following specific composition: 20 g / L oats, 0.2 g / L magnesium sulfate, 0.2 g / L potassium nitrate, 0.5 g / L dipotassium hydrogen phosphate, 20 g / L agar, balance water, pH 7.2; Preparation method: Weigh 20 g of oats, boil for 30 min, filter, add 0.2 g magnesium sulfate, 0.2 g potassium nitrate, and 0.5 g dipotassium hydrogen phosphate to the filtrate, make up to 1 L, adjust the pH to 7.2, add agar, boil, and sterilize.

[0034] The composition of the seed culture medium for *Streptomyces bluegray* was: 5 g / L glucose, 20 g / L maltodextrin, 1 g / L soluble starch, 10 g / L yeast extract, 1 g / L beef extract, 1 g / L casein peptone, with the balance being water, pH 7.2.

[0035] The composition of the fermentation medium for *Streptomyces bluegray* is: 50 g / L glucose, 25 g / L lactose, 25 g / L cottonseed meal, 4 g / L calcium carbonate, with the balance being water, pH 7.2.

[0036] The ClonExpress MultiS kit used in this invention was purchased from Novizan.

[0037] The detection method involved in this invention is as follows: Method for detecting milbemycin: 0.5 mL of fermentation broth from *Streptomyces hygroscopicus* was thoroughly mixed with 1.5 mL of ethanol, ultrasonically disrupted for 25 min, and extracted overnight. The pre-treated sample was then centrifuged at 12000 rpm for 10 min. The supernatant milbemycin extract was filtered through a 0.22 μm organic filter membrane and analyzed by liquid chromatography. The instrument used was an Agilent high-performance liquid chromatography (HPLC) system with a C18 column (Zorbax, 4.6 mm × 250 mm, 5 μm), a detection wavelength of 242 nm, an injection volume of 20 μL, and a mobile phase flow rate of 1.0 mL / min. Specific chromatographic conditions were as follows: 0-15 min: mobile phase A (methanol) eluted from 0% to 100%, mobile phase B (acetonitrile:methanol:water = 7:2:1, v / v) eluted from 100% to 0%; 15-17 min: mobile phase A maintained at 100%; 17-25 min: mobile phase B maintained at 100%. For 25-27 minutes, mobile phase A (methanol) was eluted from 100% to 0%, and mobile phase B was eluted from 0% to 100% in a gradient. Mobile phase B was maintained at 100%, and the column temperature was 28℃.

[0038] The detection method for nimocritin was as follows: 0.4 mL of fermentation broth was soaked in 1.2 mL of ethanol and extracted by rotation for 1 h. The mixture was centrifuged at 12000 rpm for 15 min, and the supernatant was collected, filtered through a 0.22 μm organic filter membrane, and then analyzed by HPLC. The HPLC conditions were: 0–30 min, 85% solvent A and 15% solvent B (solvent A: methanol; solvent B: water), flow rate of 1.0 mL / min, Agilent Zorbax SB-C18 column (Zorbax, 4.6 × 250 mm, 5 μm), and detection wavelength of 240 nm.

[0039] In this invention sbi_04891-sbi_04892 The nucleotide sequence of the gene is shown in SEQ ID NO.1; P hrdB The nucleotide sequence of the promoter is shown in SEQ ID NO.2.

[0040] SEQ ID NO.1:

[0041] SEQ ID NO.2: ccgccttccgccggaacggcggggtccgggcacgccaaacccctcctgtggctgtggccggccaccgccgtcaccttcggaccccgtggagccgctcccggttccac ggggtccgaaggtgtgatgagcaggctgcgccttcctcgcgcggccgcaaggtacgagttgatgaccttgtttatccgcatctgaccaattttgatcgcttacgggg tgtgactcgggccacgcggattgggcgtaacgctcttgggaacaacacgatgacctaagaggtgacagccgcggagggaatacggacgccgttcacggcgctgtgca tctccccggcccgcccgcaccgtcggcccattcccaagccggtggtcggcccctgtccgccgtggacggggccggaagccgtttttcaacgttccgagaggttgttc Example 1: Overexpression sbi_04891-sbi_04892 Construction and application of recombinant strains of Streptomyces hygroscopicus from Harbin In previous research, this invention obtained recombinant *Streptomyces bingchengensis* by overexpressing its own AtrA regulator-encoding gene in *Streptomyces bingchengensis* BC04. Analysis of the transcriptome data of this recombinant *Streptomyces bingchengensis* revealed that the gene encoding the ABC transporter... sbi_04891 - sbi_04892 Gene expression abundance was significantly upregulated. Therefore, this invention attempts to overexpress the gene in *Streptomyces BC04*. sbi_04891 - sbi_04892 The gene was identified and its effect on the production of milbemycin by fermentation of *Streptomyces BC04* was verified.

[0042] 1. Construction of the recombinant expression vector pSET152::sbi_04891-04892 Polymerase chain reaction (PCR) was performed using the Q5 high-fidelity enzyme system. Using the genome of *Streptomyces BC04* from Harbin as a template, primers sbi_04891-sbi_04892-LF and sbi_04891-sbi_04892-LR, PhrdB-F, and PhrdB-R were used to amplify the genome. sbi_04891-sbi_04892 Gene fragments and P hrdBPromoter fragment. The PCR reaction system was as follows: 10×KOD Buffer 5 μL, 2 mM dNTPs 5 μL, template DNA 10 pg-1000 ng, 10 μM forward and reverse primers 2 μL each, 25 mM MgSO4 2 μL, 1 U / μL KOD plus DNA polymerase 1 μL, DMSO 3 μL, ddH2O to a final volume of 50 μL. The PCR reaction conditions were as follows: 94℃ for 4 min; 94℃ for 1 min, 65℃ for 30 sec, 68℃ for 3 min, 31 cycles; 68℃ for 10 min. Xba I and Eco RI was used to double-digest plasmid pSET152 to obtain the linear vector backbone. The digestion system was: 1-2 μg plasmid pSET152, 10 μL 10×M buffer, and 10 μL BSA. Xba I 3 μL, Eco Add 3 μL of RI and bring the total volume to 100 μL with ddH2O. Use the ClonExpress MultiS kit to process the PCR-obtained... sbi_04891-sbi_04892 Gene fragments, P hrdB The promoter fragment was spliced ​​with the linear vector backbone. The splicing system was as follows: vector pSET152 60-70 ng. sbi_ 04891-sbi_04892 10 ng gene fragment, P hrdB 10 ng of promoter fragment, 2 μL of 5×CE MultiS Buffer, 1 μL of Exnase MultiS, and ddH2O were added to a final volume of 10 μL. The splicing product was then transformed into *E. coli* JM109, plated, and clones were picked and cultured in small tubes containing apramycin (final concentration 100 μg / mL). Plasmids were extracted using a plasmid extraction kit, and after electrophoresis, enzyme digestion and sequencing verification were performed to obtain the recombinant plasmid pSET152::sbi_04891-04892 (a schematic diagram of the construction of this recombinant plasmid is shown below). Figure 1 (As shown).

[0043] 2. Construction of recombinant strain BC04 / Osbi_04891-04892 The recombinant plasmid pSET152::sbi_04891-04892 was transformed into *Escherichia coli* ET12567 / pUZ8002, and then introduced into *Streptomyces BC04* via conjugation transfer. The conjugates were then picked onto SKYM medium containing apramycin (final concentration 16 μg / mL) and nalidixic acid (final concentration 25 μg / mL) and cultured at 28°C for 9 days. The results were then validated. sbi_04891-sbi_ 04892Gene transcriptional expression levels are overexpressed. sbi_04891-sbi_04892 The recombinant strain BC04 / Osbi_04891-04892.

[0044] 3. Application of recombinant strain BC04 / Osbi_04891-04892 in the fermentation production of milbemycin The recombinant strain BC04 / Osbi_04891-04892, the control strain BC04 / pSET152, and the original strain BC04 were inoculated onto SKYM solid medium and cultured at 28°C for 9 days. Spores of about 1 square centimeter were scraped off and inoculated into the seed culture medium of *Streptomyces hygroscopicus*, and cultured at 28°C and 250 rpm for 46 hours. Then, at an inoculation rate of 6%, the inoculated broth was added to the fermentation medium of *Streptomyces hygroscopicus*, and cultured at 28°C and 250 rpm for 9 days to obtain the fermentation broths of strains BC04 / Osbi_04891-04892, BC04 / pSET152, and BC04, respectively.

[0045] The control strain BC04 / pSET152 was constructed as follows: the empty plasmid pSET152 was transformed into *Escherichia coli* ET12567 / pUZ8002, and then introduced into *Streptomyces cerevisiae* BC04 via conjugation transfer. The conjugate was then picked onto SKYM medium containing apramycin (final concentration 16 μg / mL) and nalidixin (final concentration 25 μg / mL), and cultured at 28°C for 9 days to obtain the control strain BC04 / pSET152.

[0046] The content of milbemycin in the fermentation broth of strains BC04 / Osbi_04891-04892, BC04 / pSET152, and BC04 was determined by HPLC. The results are as follows: Figure 2 As shown in the figure, the milbemycin yield of the recombinant strain BC04 / Osbi_04891-04892 (3900.27 mg / L) was approximately 0.5 times higher than that of the control strain BC04 / pSET152 (2531 mg / L). This indicates that the overexpression of the efflux gene... sbi_04891-sbi_04892 The recombinant strain can be used to increase the yield of milbemycin produced by fermentation of Streptomyces BC04 from Harbin.

[0047] Example 2: Overexpression sbi_04891-sbi_04892 Construction and application of recombinant Streptomyces bluegray strains 1. Construction of recombinant strain NMWT1 / Osbi_04891-sbi_04892 The recombinant plasmid pSET152::sbi_04891-04892 obtained in step 1 of Example 1 was transformed into Escherichia coli ET12567 / pUZ8002, and then introduced into Streptomyces blue-gray toxin NMWT1 via conjugation transfer. The conjugates were then picked onto IPS3 medium containing apramycin (final concentration 16 μg / mL) and nalidixic acid (final concentration 25 μg / mL), and cultured at 28°C for 9 days. The results were then verified. sbi_04891-sbi_04892 Gene transcriptional expression levels are overexpressed. sbi_04891-sbi_04892 The recombinant strain NMWT1 / Osbi_04891-sbi_04892.

[0048] 2. Application of recombinant strain NMWT1 / Osbi_04891-sbi_04892 in the fermentation production of nimocriptine. The recombinant strain NMWT1 / Osbi_04891-sbi_04892, the control strain NMWT1 / pSET152, and the original strain NMWT1 were inoculated onto ISP3 solid medium and cultured at 37°C for 9 days. Spores were then collected and a spore suspension was prepared. The absorbance of the spore suspension was measured at 450 nm using a UV spectrophotometer, and the spore suspension was diluted to an OD value of [missing value]. 450 The value is approximately 0.2; 1 mL of OD was drawn. 450 A spore suspension at 0.2 g / L was inoculated into a 250 mL Erlenmeyer flask containing 25 mL of seed culture medium, and then cultured in a shaker at 28°C and 250 rpm for 46 hours. When the seed culture matured, 1.8 mL of the seed culture was transferred to a 250 mL Erlenmeyer flask containing 30 mL of fermentation culture medium and cultured in a shaker at 28°C and 250 rpm for another 9 days to obtain the fermentation broths of strains NMWT1 / Osbi_04891-sbi_04892, NMWT1 / pSET152, and NMWT1, respectively.

[0049] The control strain NMWT1 / pSET152 was constructed as follows: the empty plasmid pSET152 was transformed into *Escherichia coli* ET12567 / pUZ8002, and then introduced into *Streptomyces bluegrass* NMWT1 via conjugation transfer. The conjugate was then picked onto IPS3 medium containing apramycin (final concentration 16 μg / mL) and nalidixin (final concentration 25 μg / mL), and cultured at 28°C for 9 days to obtain the control strain NMWT1 / pSET152.

[0050] The content of nimocritin in the fermentation broth of strains NMWT1 / Osbi_04891-sbi_04892, NMWT1 / pSET152, and NMWT1 was determined by HPLC. The results are as follows: Figure 3As shown in the figure, the nimocriptine yield of the recombinant strain NMWT1 / Osbi_04891-sbi_04892 (672.31 mg / L) was approximately double that of the control strain NMWT1 / pSET152 (310.12 mg / L). This indicates that the overexpression of the efflux gene... sbi_04891-sbi_04892 The recombinant strain can be used to increase the yield of nimodicin produced by fermentation of Streptomyces bluegrass NMWT1.

[0051] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. An efflux gene sbi_04891-sbi_04892 Its application in increasing the yield of macrolide antibiotics by Streptomyces is characterized by... The efflux gene sbi_04891-sbi_04892 The nucleotide sequence is shown in SEQ ID NO.1, and the macrolide antibiotic is milbemycin or nimocritin.

2. The application according to claim 1, characterized in that, The application is in the case of Streptomyces glaciers in Harbin ( Streptomyces bingchenggensis Overexpression in BC04 sbi_04891-sbi_04892 Genes, or in Streptomyces bluegrayense ( Streptomyces cyaneogriseus Overexpression in NMWT1 sbi_04891-sbi_04892 Gene.

3. A device containing the efflux gene as described in claim 1 sbi_04891-sbi_04892 Application of recombinant vectors in increasing the yield of macrolide antibiotics by Streptomyces.

4. The application according to claim 3, characterized in that, The application involves introducing the recombinant vector into Escherichia coli and then transferring it into Streptomyces BC04 or Streptomyces NMWT1.

5. The application according to claim 3, characterized in that, The method for constructing the recombinant vector includes the following steps: double digestion of plasmid pSET152 to obtain a linear vector backbone; using the genome of *Streptomyces BC04* from Harbin as a template, amplification... sbi_04891-sbi_04892 Gene fragments and P hrdB Promoter fragment; linking the linear vector skeleton with sbi_04891-sbi_ 04892 Gene fragments and P hrdB The promoter fragments are spliced ​​together to obtain the recombinant vector; the P hrdB The nucleotide sequence of the promoter is shown in SEQ ID NO.

2.

6. The application according to claim 5, characterized in that, The amplification sbi_04891-sbi_04892 The nucleotide sequences of the upstream and downstream primers used for the gene fragment are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively, for amplifying P. hrdB The nucleotide sequences of the upstream and downstream primers used for the promoter fragment are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

7. The application according to claim 5, characterized in that, The restriction endonuclease used in the double digestion is: Xba I and Eco RI.

8. A device comprising the contents of claim 1 sbi_04891-sbi_04892 The use of the gene or the recombinant strain of the recombinant vector described in claim 3 in increasing the production of macrolide antibiotics by Streptomyces.

9. The application according to claim 8, characterized in that, The method for constructing the recombinant bacteria includes the following steps: constructing the recombinant bacteria described in claim 1. sbi_04891-sbi_04892 The gene overexpression integration plasmid was transformed into E. coli, and then the overexpression integration plasmid was introduced into the starting strain through intergeneric fusion transfer experiment to obtain recombinant bacteria.

10. The application according to claim 9, characterized in that, The starting strain is either *Streptomyces berberis* BC04 or *Streptomyces bluegrayense* NMWT1.

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