Streptomycete engineering bacterium with high yield of josamycin as well as construction method and application of streptomycete engineering bacterium

Abstract

The invention relates to a streptomyces engineering bacterium with high yield of josamycin as well as a construction method and application of the streptomyces engineering bacterium. The engineering bacterium is a high-yield strain which is obtained by taking Streptomyces nanonensis ATCC17835 as an original strain and carrying out over-expression on regulatory protein genes JosR1 and JosR2 in a genome of the Streptomyces nanonensis ATCC17835, and the engineering bacterium is named as Streptomyces nanonensis CBR18-P001 (the preservation number is CCTCC No. M 2025197). The invention further discloses a preparation method of the engineering bacterium, and a preparation method of the engineering bacterium comprises the following steps: taking the Streptomyces nanonensis ATCC17835 as the original strain, carrying out over-expression on the regulatory protein genes JosR1 and JosR2 in the genome of the engineering bacterium. The fermentation yield of the strain josamycin can reach 7000 mg / L or above, and a fermentation product contains a characteristic impurity O (the molecular formula and molecular weight are 825, the relative retention time of a high performance liquid phase is 0.95, and the mass spectrum characteristic peak m / z is 826 [M + H]) as a biomarker. The invention also provides a construction method of the strain, a fermentation production process and an identification method based on characteristic impurities.

Description

Technical Field

[0001] This invention belongs to the fields of synthetic biology and microbial fermentation technology, specifically relating to a high-yield Josamycin-producing engineered Streptomyces strain, its construction method, and its application. Background Technology

[0002] Josamycin was first discovered by Yamanouchi Pharmaceutical Co., Ltd. in Japan. It is produced by fermentation of the josamycin variant of Streptomyces narbonensis (Streptomyces narbonensis var. josamyceticus). Josamycin inhibits the synthesis of pathogen proteins and has a significant inhibitory effect on Gram-positive bacteria, some Gram-negative bacteria, and pathogens such as mycoplasma and chlamydia.

[0003] Currently, this product is exclusively produced by Astellas Pharma in Japan, while China imports tens of tons of josamycin and its semi-synthetic product, josamycin propionate, raw material, annually. For decades, domestic production of this product has been lacking, primarily due to low fermentation unit density, resulting in uncompetitive production costs. Furthermore, effectively tracking and identifying specific high-yield engineered strains in industrial production to prevent strain loss and infringement remains a pressing issue.

[0004] There are two main methods for increasing josamycin yield: traditional mutagenesis and selection based on physicochemical methods, and molecular breeding based on synthetic biology and gene editing technologies. Traditional mutagenesis and screening methods (such as ultraviolet and chemical mutagenesis) are highly unpredictable and time-consuming. With the rapid development of synthetic biology and gene editing technologies, rationally modifying microbial hosts through genetic engineering has become a core strategy for breaking through antibiotic production bottlenecks and developing innovative drugs.

[0005] The core of the biosynthetic gene cluster of josamycin comprises four main categories: polyketide synthase (PKS) genes, post-modification genes, regulatory genes, and resistance genes. The polyketide synthase gene consists of multiple modules, each corresponding to a step in the synthesis of the polyketide chain, assembling the macrocyclic lactone skeleton of josamycin through steps such as condensation, reduction, and dehydration. Post-modification genes are responsible for modifying the skeleton through glycosylation, methylation, etc., determining its antibacterial activity and specificity. Regulatory genes regulate the synthesis process of josamycin by activating or inhibiting the expression of PKS genes and post-modification genes. Resistance genes encode proteins that enable the bacteria to tolerate josamycin, preventing them from being killed by the synthesized antibiotic. In the prior art, patent document CN114517175A discloses a genetically engineered bacterium that increases yield by approximately 15-23% by overexpressing specific polyacrylamide synthesis elongation and modification-related genes, and systematically identifies a biosynthetic gene cluster containing 32 open reading frames, also indicating the existence of genes related to biosynthetic regulation. However, existing technologies (including CN114517175A) mainly involve the modification of structural genes such as polyketide synthase and post-modification enzymes. They do not provide clear technical insights or successful implementation plans on how to achieve high industrial yields by rationally modifying the two key regulatory genes, JosR1 and JosR2, especially the impact of simultaneously modifying them on yield and metabolome.

[0006] During their in-depth research, the applicant discovered that the functional expression of JosR1 and JosR2 in the complex biosynthetic regulatory network of josamycin is not a simple parallel or additive relationship. Through systematic combinatorial screening experiments, the simultaneous overexpression of JosR1 and JosR2 was attempted. Unexpectedly, the engineered bacteria not only achieved a significant increase in josamycin production but also produced a structurally distinct characteristic impurity O that is not present in the original active pharmaceutical ingredient (API) of josamycin. This correlation between "high production" and the simultaneous appearance of a "new metabolic marker" is unprecedented and unpredictable in existing technologies. Summary of the Invention

[0007] To address the problems of low yield, insufficient genetic stability, and poor industrial adaptability of existing josamycin-producing strains, this invention provides a high-yielding, genetically stable Streptomyces strain with unique metabolic markers, as well as its construction method and applications.

[0008] This invention modifies the starting strain Streptomyces nabothianum using synthetic biotechnology, overexpressing the regulatory protein genes JosR1 and JosR2, to construct an engineered strain that produces high levels of josamycin and carries characteristic biomarkers.

[0009] This invention provides a high-yield josamycin-producing engineered Streptomyces strain, which is obtained by overexpressing the regulatory protein genes JosR1 and JosR2, starting with Streptomyces narbonensis ATCC17835.

[0010] Preferably, the overexpression of the regulatory protein genes JosR1 and JosR2 described in this invention can be achieved through various gene manipulation strategies known in the art. For example, the expression of these genes can be driven by strong constitutive promoters or inducible promoters, with expression controlled by independent promoters. The gene expression cassette used for overexpression can be integrated into a specific site on the host bacterial chromosome (e.g., through homologous recombination) or constructed on a plasmid capable of autonomous replication in the host bacterial cell.

[0011] The coding sequence of the regulatory protein gene JosR1 is shown in SEQ ID NO.4, and the coding sequence of the regulatory protein gene JosR2 is shown in SEQ ID NO.5.

[0012] Preferably, the engineered Streptomyces strain is named (Streptomyces narbonensis) CBR18-P001, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC No. M 2025197, deposit date: February 11, 2025, and deposit address: Wuhan University, Wuhan, China.

[0013] This invention also provides a method for constructing the engineered Streptomyces strain, which involves overexpressing the regulatory proteins JosR1 and JosR2 in the ATCC17835 genome using homologous recombination technology to obtain the engineered Streptomyces strain, comprising the following steps: 1. Using the genomic DNA of the starting strain ATCC17835 as a template, amplification was performed using primers JosR1R2-F (sequence shown in SEQ ID NO.1) and JosR1R2-R (sequence shown in SEQ ID NO.2) to obtain a DNA fragment R1R2 containing the JosR1 and JosR2 genes, the sequence of which is shown in SEQ ID NO.3; 2. The DNA fragment R1R2 was ligated to the plasmid pSET152HK to construct the overexpression vector pSET152HK-R1R2A; 3. The overexpression vector pSET152HK-R1R2A was transformed into competent cells and introduced into the starting strain ATCC17835 via conjugation transfer. After culturing and screening on a medium containing aspirin, the engineered Streptomyces strain was obtained.

[0014] The present invention also provides a method for producing josamycin, comprising the following steps: fermenting the engineered Streptomyces strain and isolating josamycin from the fermentation product.

[0015] The fermentation product contains a substance with the molecular formula: A josamycin derivative with a molecular weight of 825, a high-performance liquid chromatography relative retention time of 0.95, and a mass spectrometry characteristic peak of m / z 826 [M+H]⁺ was identified as a characteristic impurity O.

[0016] The fermentation culture medium preferably contains the following components: 80 g / L soybean oil, 24 g / L corn gluten meal, 12 g / L sesame cake powder, 8 g / L L-leucine, 0.3 g / L manganese sulfate, 0.3 g / L magnesium sulfate, and 0.3 g / L ferrous sulfate, and the pH value of the fermentation culture medium before sterilization is 7.5.

[0017] The preferred fermentation conditions are: a culture temperature of 28-32℃, a dissolved oxygen level of not less than 20% during the culture process, and a culture time of 108-192 hours; and the yield of josamycin in the engineered Streptomyces strain during fermentation is higher than 7000 mg / L.

[0018] The present invention also provides a method for identifying the engineered bacteria or its fermentation product, comprising the following steps: (1) Obtain the sample to be tested; (2) Analyze the metabolite profile of the sample to be tested; (3) Detect whether the metabolite profile contains molecules with the following molecular formula: The characteristic impurity O is [M+H]⁺, with a molecular weight of 825, a relative retention time of 0.95 in high-performance liquid chromatography, and a characteristic peak at m / z 826 in mass spectrometry. (4) If the characteristic impurity O is detected, the sample to be tested is determined to be derived from or contain the engineered Streptomyces strain as described above.

[0019] The detection described above is performed using high-performance liquid chromatography (HPLC), and the chromatographic conditions include: Mobile phase A is a mixture of 30 g / L sodium perchlorate solution and acetonitrile-tetrahydrofuran mixture, wherein the volume ratio of acetonitrile to tetrahydrofuran is 70:30, and the volume ratio of 30 g / L sodium perchlorate solution to the acetonitrile-tetrahydrofuran mixture is 98:2. Mobile phase B is a mixture of acetonitrile and tetrahydrofuran in a volume ratio of 98:2; The detection wavelength is 231 nm; The retention time of characteristic impurity O relative to josamycin is 0.95.

[0020] The engineered Streptomyces strain provided by this invention or the josamycin obtained by the production method described above can be used to prepare drugs for treating infections.

[0021] This invention significantly increases the final yield of josamycin by simultaneously overexpressing JosR1 and JosR2, accompanied by the generation of new metabolic characteristics. This technical solution achieves both a breakthrough in yield and specific changes in the metabolic profile. Its overall technical effect cannot be obviously derived from existing technologies, but is based on a deep understanding of the interactions of regulatory networks and the application of specific technical means.

[0022] The beneficial effects of this invention are as follows: 1. Significantly increased yield: The josamycin yield of the engineered Streptomyces strain CBR18-P001 provided by this invention is 32 times that of the original strain, and the fermentation level can reach more than 7000 mg / L. This increase far exceeds the closest existing technology (such as the conventional increase of about 15-23% obtained by modifying a single modified enzyme gene in CN114517175A), indicating that this invention adopts a completely different and more efficient technical path.

[0023] 2. Possesses unique biomarkers: While achieving high yield, the fermentation product of the engineered Streptomyces strain CBR18-P001 of this invention contains characteristic impurity O, which can be used as a specific marker for strains and products for traceability and infringement determination.

[0024] 3. Good genetic stability: The core high-yield strain CBR18-P001 achieves gene overexpression through chromosome integration, without the need for antibiotic stress maintenance, and has good passage stability.

[0025] 4. Good prospects for industrial application: The fermentation process is relatively simple, the yield is high, and it is highly compatible with existing production processes. Attached Figure Description

[0026] The accompanying drawings of this invention are described below. Figure 1 Schematic diagram of the structure of the overexpression vector pSET152HK-R1R2A Figure 2 Colony morphology of engineered Streptomyces CBR18-P001 on ISP3 solid medium Figure 3 Comparison of yields of engineered Streptomyces strain CBR18-P001 and the original strain ATCC17835 after 120 hours of shake-flask fermentation Figure 4 Yield variation graph of three batches of engineered Streptomyces strain CBR18-P001 fermented continuously in a 20t fermenter. Figure 5Comparison of high-performance liquid chromatograms of fermentation products of engineered Streptomyces strain CBR18-P001(A) and original API of josamycin Figure 6 Characteristic impurity O ( ) structural diagram Figure 7 Characteristic impurity O ( LC-MS results spectrum Detailed Implementation

[0027] The following detailed description provides specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, experimental methods in the following examples are generally performed according to conventional methods and conditions, in accordance with the techniques or conditions described in the literature in the art, or according to the product manual.

[0028] Unless otherwise specified, all materials and reagents used in the following implementation examples are commercially available.

[0029] Culture medium used in the examples MS medium Mannitol 20 g / L, soybean flour 20 g / L, agar 20 g / L, add distilled water to make up to 1 L, sterilize at 115℃ for 30 min.

[0030] 2×YT medium Add 16 g / L Tryptone, 10 g / L Yeast Extract, and 5 g / L NaCl to distilled water to a final volume of 1 L, then sterilize at 121°C for 30 min.

[0031] ISP3 solid culture medium 20 g / L oat flour, 0.001 g / L ferric sulfate heptahydrate, 0.001 g / L manganese chloride, 0.001 g / L zinc sulfate, 18 g / L agar, add distilled water to bring the volume to 1 L, adjust the pH to 7.3±0.2, and sterilize at 121 ℃ for 30 minutes.

[0032] Seed culture medium Soybean meal powder 5 g / L, yeast extract powder 5 g / L, corn steep liquor powder 5 g / L, corn gluten powder 5 g / L, soybean oil 5 g / L, calcium carbonate 3 g / L. Prepared with drinking water, pH adjusted to 6.8 before sterilization. Sterilize at 121 ℃ for 30 minutes.

[0033] Fermentation medium Soybean oil 80 g / L, corn gluten meal 24 g / L, sesame cake powder 12 g / L, L-leucine 8 g / L, manganese sulfate 0.3 g / L, magnesium sulfate 0.3 g / L, ferrous sulfate 0.3 g / L. Prepared as drinking water, pH adjusted to 7.5 before sterilization. Sterilize at 121 ℃ for 30 minutes.

[0034] Example 1: Construction method of Streptomyces strain CBR18-P001 with high josamycin production Using genomic DNA from the *Streptomyces narbonensis* ATCC17835 strain purchased from Guangzhou Yuechen Biotechnology Co., Ltd. as a template, a 3.1 kb DNA fragment R1R2 (SEQ ID NO.3) containing both the JosR1 and JosR2 regulatory genes was amplified using primer pairs JosR1R2-F (SEQ ID NO.1) and JosR1R2-R (SEQ ID NO.2). The amplified fragment R1R2 was digested with NdeI and ligated into the NdeI-digested pSET152HK plasmid to obtain the overexpression vector pSET152HK-R1R2A. Sequencing confirmed its correctness. Figure 1 As shown.

[0035] The plasmid pSET152HK-R1R2A was transformed into E. coli ET12567 / pUZ8002 competent cells. The cells were plated on LB agar plates containing the corresponding antibiotic and incubated overnight at 37°C. Single colonies were picked and inoculated into liquid LB medium containing the corresponding antibiotic and incubated overnight at 37°C. The overnight culture was then inoculated at a 1:100 ratio into 10 mL of liquid LB medium containing the corresponding antibiotic and incubated at 37°C until OD600 = 0.4–0.6. The cells were centrifuged, collected, washed twice with an equal volume of liquid LB medium, and resuspended in 500 μL of liquid LB medium for later use.

[0036] Mycelia of the starting strain ATCC17835 were collected using 2×YT medium, and the final mycelial suspension volume was adjusted to 500 μL. 500 μL of Escherichia coli cells were mixed with 500 μL of Streptomyces mycelia and spread onto MS solid medium containing 10 mg Cl2, and cultured at 28°C. After culturing for 16-20 h, nadolol acid (final concentration 25 μg / mL) and apramycin (final concentration 20 μg / mL) were evenly spread onto the surface of the aforementioned solid medium containing Streptomyces and Escherichia coli, and cultured at 28°C for another 5-7 days.

[0037] The resulting Streptomyces colonies were transferred to MS solid medium containing apramycin (final concentration 20 μg / mL) for expansion and validation. A high-yielding strain was obtained from the transformants and named *Streptomyces narbonensis* CBR18-P001. The morphology of the strain cultured on ISP3 solid medium at 30°C for 7 days was as follows: Figure 2 As shown.

[0038] Example 2: Shake-flask validation of josamycin yield from engineered Streptomyces strain CBR18-P001 a. The engineered Streptomyces strain CBR18-P001 constructed in Example 1 and the starting strain ATCC17835 were cultured in a shake flask fermentation medium: the shake flask fermentation conditions were 30℃, 220 r / min, and 120 h. b. Josamycin was isolated from the fermentation broth.

[0039] c. Sample processing procedure: Take 2 ml of fermentation broth, place it in a 50 mL centrifuge tube, add 18 mL of anhydrous ethanol, mix well, sonicate for 30 minutes, shake well, take an appropriate amount of sample solution, place it in a high-speed centrifuge, centrifuge at 20000 rpm for 1 minute, take it out, take the supernatant and filter it through a 0.22 μm filter membrane, use the filtrate as the test solution, and determine its yield by HPLC.

[0040] d. The parameters for liquid chromatography determination of josamycin yield are as follows: Column: Octadecylsilane-bonded silica gel as packing material (YMC, J'Sphere ODS-H80, 4.6 mm × 250 mm, 4 μm); Detection wavelength: 231 nm; Flow rate: 1.0 mL / min; Mobile phase: Mobile phase A is a mixture of 30 g / L sodium perchlorate solution (pH adjusted to 2.5 with 1 mol / L hydrochloric acid solution) and acetonitrile-tetrahydrofuran mixture, wherein the volume ratio of acetonitrile to tetrahydrofuran is 70:30, and the volume ratio of 30 g / L sodium perchlorate solution to the acetonitrile-tetrahydrofuran mixture is 98:2; Mobile phase B: Acetonitrile-tetrahydrofuran (98:2) (V / V); Perform gradient elution according to the table below: Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 30 71 29 38 71 29 40 21 79 55 21 79 55.01 100 0 60 100 0 Figure 3 This is a schematic diagram comparing the yields of the engineered Streptomyces strain CBR18-P001 and the original strain ATCC 17835 during shake-flask fermentation. After 120 hours of fermentation, the josamycin yield of the engineered Streptomyces strain was 7456 mg / L, which was 32 times that of the original strain.

[0041] Example 3: Scale Validation of Engineered Streptomyces CBR18-P001 Fermenter

[0042] The engineered Streptomyces strain CBR18-P001 was fermented in a 20T fermenter using the same fermentation medium as in Example 2. The culture conditions were 30 °C, dissolved oxygen ≥20%, and fermentation time was 119 h. Three consecutive batches of fermentation were carried out. Figure 4 This is a schematic diagram showing the change in josamycin production. The average production of josamycin can reach 7205 mg / L.

[0043] Example 4: Detection of bacterial strain markers

[0044] A sample of the CBR18-P001 fermentation broth, obtained after 120 hours of shake-flask fermentation in Example 2, was analyzed using the liquid chromatography method described in Example 2. Figure 5 As shown, a josamycin derivative containing the characteristic impurity O was detected in the product of the engineered Streptomyces strain CBR18-P001, while this substance was not present in the original josamycin API. This demonstrates that the josamycin derivative containing the characteristic impurity O can serve as a marker for the engineered Streptomyces strain CBR18-P001. The molecular formula of the characteristic impurity O was identified through analysis. The molecular weight is 825, the relative retention time is 0.95, and the structural formula is as follows: Figure 6 As shown.

[0045] LC-MS verification: Electrospray ionization source, positive ion mode, scan range m / z 50-2000, confirming the molecular weight of the characteristic impurity O. The molecular weight of the compound corresponding to the characteristic peak was identified as 825 (m / z 826 [M+H]⁺), as shown in the results. Figure 7 As shown.

[0046] Example 5: Application of strain markers in identification

[0047] Metabolites were extracted from fermentation samples or commercial products suspected of containing the engineered bacteria of this invention, and analyzed by high-performance liquid chromatography (HPLC) or LC-MS. If the sample contained the characteristic impurity O, it could be highly certain that the sample originated from or was adulterated with the engineered bacteria of this invention, which could be used for intellectual property infringement determination.

[0048] The above embodiments demonstrate that this invention has successfully constructed a high-yielding engineered strain of josamycin with the characteristic impurity O label. Using this engineered strain in a 20t fermenter, the yield of josamycin can reach over 7000 mg / L for industrial production. This engineered strain has been successfully applied to continuous production in fermenters of 60 tons and above, and in over 80 production batches, the josamycin yield has remained consistently above 7000 mg / L, proving its excellent industrial scalability and production stability.

[0049] Explanation of gene expression patterns

[0050] In this invention, the expression of the JosR1 and JosR2 genes is not limited to a specific method, and those skilled in the art can choose a suitable expression system as needed. Expression can be driven by strong constitutive promoters or inducible promoters, and can be controlled by independent promoters. The expression can be integrated into specific chromosomal sites or exist on autonomously replicating plasmids.

[0051] Instructions for drug use

[0052] The josamycin produced by the engineered bacteria of this invention can be used to prepare anti-infective drugs. The specific drug dosage form and preparation method can be adjusted according to clinical needs.

[0053] The sequences used in the embodiments of this invention are as follows: Serial identifier Length (bp) type Sequence Description SEQ ID NO.1 32 Primers JosR1R2-F (primers used to amplify the JosR1 and JosR2 genes) SEQ ID NO.2 31 Primers JosR1R2-R (primers used to amplify the JosR1 and JosR2 genes) SEQ ID NO.3 3185 DNA fragments R1R2 fragment containing the JosR1 and JosR2 genes SEQ ID NO.4 1953 gene coding sequence JosR1 gene coding sequence SEQ ID NO.5 1173 gene coding sequence JosR2 gene coding sequence The sequence information mentioned in this specification can be found in the electronic sequence list file that was filed on the same day as this application and complies with the requirements of the Patent Office, the contents of which are incorporated herein by reference.

Claims

1. A high-yield streptomycin-producing engineered strain, characterized in that, The strain was *Streptomyces naboides* obtained by overexpressing the regulatory protein genes JosR1 and JosR2. Streptomyces narbonensis Engineered bacteria; wherein the coding sequence of the regulatory protein gene JosR1 is shown in SEQ ID NO.4, and the coding sequence of the regulatory protein gene JosR2 is shown in SEQ ID NO.

5.

2. The engineered Streptomyces strain according to claim 1, characterized in that, The strain was obtained from the strain with accession number CCTCCNo. M 2025197. Streptomyces narbonensis CBR18-P001 represents.

3. A method for constructing an engineered Streptomyces strain according to claim 1 or 2, characterized in that, The engineered Streptomyces strain was obtained by overexpressing the regulatory proteins JosR1 and JosR2 in the genome of ATCC17835 using homologous recombination technology.

4. The construction method according to claim 3, characterized in that, Includes the following steps: (1) Using the genomic DNA of the starting strain ATCC17835 as a template, amplification was performed using primers JosR1R2-F (sequence shown in SEQ ID NO.1) and JosR1R2-R (sequence shown in SEQ ID NO.2) to obtain a DNA fragment R1R2 containing the JosR1 and JosR2 genes. The sequence of the DNA fragment R1R2 is shown in SEQ ID NO.

3. (2) The DNA fragment R1R2 was ligated to the plasmid pSET152HK to construct the overexpression vector pSET152HK-R1R2A; (3) The overexpression vector pSET152HK-R1R2A was transformed into competent cells and introduced into the starting strain ATCC17835 by conjugation transfer. After culturing and screening on a medium containing aspirin, the engineered Streptomyces strain was obtained.

5. A method for producing josamycin, characterized in that, Includes the following steps: The *Streptomyces* strain described in claim 1 or 2 is used for fermentation culture, and josamycin is isolated from the fermentation product; wherein the fermentation product contains a substance with the molecular formula […]. A josamycin derivative with a molecular weight of 825, a relative retention time of 0.95 in high-performance liquid chromatography, and a characteristic peak of 826 [M+H]⁺ in mass spectrometry was identified as a characteristic impurity O.

6. The method for producing josamycin according to claim 5, characterized in that, The fermentation medium used in the fermentation culture contains the following components: 80 g / L soybean oil, 24 g / L corn gluten meal, 12 g / L sesame cake powder, 8 g / L L-leucine, 0.3 g / L manganese sulfate, 0.3 g / L magnesium sulfate, and 0.3 g / L ferrous sulfate. The pH value of the fermentation medium before sterilization is 7.

5.

7. The method for producing josamycin according to claim 5 or 6, characterized in that, The fermentation conditions are as follows: the culture temperature is 28~32℃, the dissolved oxygen level is not lower than 20% during the culture process, and the culture time is 108~192 hours; and the yield of josamycin in the engineered Streptomyces strain during the fermentation culture is higher than 7000 mg / L.

8. A method for identifying the engineered Streptomyces strain according to claim 1 or 2, or the fermentation product obtained by the method according to any one of claims 5 to 7, characterized in that, Includes the following steps: (1) Obtain the sample to be tested; (2) Analyze the metabolite profile of the sample to be tested; (3) Detect whether the metabolite profile contains molecules with the following molecular formula: The molecular weight is 825, the relative retention time in high performance liquid chromatography is 0.95, the characteristic peak in mass spectrometry is 826 m / z, and the characteristic impurity O of [M+H]⁺ is present. (4) If the characteristic impurity O is detected, the sample to be tested is determined to be derived from or contain the engineered Streptomyces strain described in claim 1 or 2.

9. The identification method according to claim 8, characterized in that, The detection was performed using high-performance liquid chromatography (HPLC), with the following chromatographic conditions: Mobile phase A is a mixture of 30 g / L sodium perchlorate solution and acetonitrile-tetrahydrofuran mixture, wherein the volume ratio of acetonitrile to tetrahydrofuran is 70:30, and the volume ratio of 30 g / L sodium perchlorate solution to the acetonitrile-tetrahydrofuran mixture is 98:

2. Mobile phase B is a mixture of acetonitrile and tetrahydrofuran in a volume ratio of 98:2; The detection wavelength is 231 nm; The retention time of characteristic impurity O relative to josamycin is 0.

95.

10. The use of the engineered Streptomyces strain according to claim 1 or 2 in the preparation of a medicament for treating infections.

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