Streptomyces gilvosporeus engineering strain with high yield of natamycin and construction method of streptomyces gilvosporeus engineering strain

Abstract

The invention discloses a streptomyces gilvosporeus engineering strain for high yield of natamycin and a construction method of the streptomyces gilvosporeus engineering strain, and belongs to the technical field of industrial microorganisms. According to the invention, the streptomyces gilvosporeus Z1403 strain with increased natamycin yield and good passage stability is obtained through screening, and the strain is taken as a chassis cell to co-express epwhiG and dasR genes, so that an engineering strain with increased natamycin yield is constructed. The yield of natamycin obtained through shake-flask fermentation of the engineering strain is increased by 64.7% compared with that of an original strain; the yield in a 5L fermentation tank can be 13.5 g.L <-1 >, and the strain has certain industrial application potential.

Description

Technical Field

[0001] This invention relates to an engineered strain of Streptomyces chrysogenum that produces high levels of natamycin and its construction method, belonging to the field of industrial microbial technology. Background Technology

[0003] Natamycin, also known as pimozycin, is mainly produced by Streptomyces species such as *Streptomyces natalensis*, *Streptomyces chattanoogensis*, and *Streptomyces gilvosporeus*. Improving the synthetic capacity of producing strains is an effective way to reduce the production cost of natamycin. Traditional mutagenesis (including physical and chemical mutagenesis) is widely used in the selection of high-yielding natamycin strains due to its simplicity and the fact that it does not require in-depth knowledge of the microbial genetic background. However, this method suffers from low iteration efficiency, high mutation randomness, and long screening cycles, making it difficult to achieve continuous and stable improvement in secondary metabolites. Even with a large number of mutants, it is often difficult to quickly identify and obtain truly high-yielding dominant strains. With further research into the natamycin synthesis mechanism, genetic engineering breeding has become a new strategy for improving natamycin production. However, genetic engineering regulation methods usually focus on strengthening or inhibiting specific metabolic pathways. Since the genetic background of natamycin biosynthesis is not clear at present, how to select appropriate genetic engineering modification sites remains one of the difficulties in improving natamycin production. Summary of the Invention

[0004] To improve the synthesis efficiency of natamycin, this invention aims to provide a genetic engineering method for constructing a high-natamycin-producing *Streptomyces gilvosporeus*. This method significantly enhances the natamycin synthesis capacity of *Streptomyces gilvosporeus* by utilizing transcriptional machinery engineering and co-expressing key transcription factors, thereby effectively increasing natamycin yield.

[0005] This invention provides a strain of Streptomyces gilvosporeus Z1403, which was deposited on May 13, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with the deposit number CGMCC No. 34513. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0006] This invention also provides the application of the epwhiG gene in increasing the natamycin yield of engineered Streptomyces chrysogenum.

[0007] In one embodiment, the nucleotide sequence of the epwhiG gene is shown in SEQ ID NO.2.

[0008] In one embodiment, the application involves expressing the epwhiG gene in Streptomyces chrysophagus.

[0009] In one embodiment, the application involves co-expressing the epwhiG gene and the global transcription factor dasR in Streptomyces chrysosporium.

[0010] In one embodiment, the *Streptomyces chrysogenum* includes, but is not limited to, *Streptomyces chrysogenum* Z1403.

[0011] This invention provides an engineered strain of Streptomyces chrysogenum that expresses the epwhiG gene and the global transcription factor dasR.

[0012] In one embodiment, the nucleotide sequence of the epwhiG gene is shown in SEQ ID NO.2; and the nucleotide sequence of the dasR gene is shown in SEQ ID NO.3.

[0013] In one embodiment, the engineered Streptomyces chrysosporium strain is Streptomyces chrysosporium Z1403 as the starting strain.

[0014] In one embodiment, the expression of the epwhiG gene and the global transcription factor dasR is regulated by a promoter with a nucleotide sequence as shown in SEQ ID NO.6.

[0015] In one embodiment, the nucleotide sequence of the promoter-containing gene expression fragment PermE*-epwhiG is shown in SEQ ID NO.5.

[0016] The present invention also provides a method for increasing the yield of natamycin from *Streptomyces thuringiensis*, comprising the following steps:

[0017] (1) Construction of recombinant plasmid pIB139-epwhiG-dasR: The dasR gene, T0 fragment, and Perm-epwhiG fragment were inserted into the multiple cloning site of the pIB139 vector plasmid to obtain the recombinant plasmid pIB139-epwhiG-dasR.

[0018] (2) Preparation of E. coli ET12567 containing recombinant plasmid pIB139-epwhiG-dasR: The recombinant plasmid pIB139-epwhiG-dasR obtained in step (4) was transformed into E. coli DH5α. After the transformants grew, colony PCR was performed for verification. After verification, the transformants were introduced into E. coli ET12567 to obtain E. coli ET12567 containing recombinant plasmid pIB139-epwhiG-dasR.

[0019] (3) Preparation of the genetically engineered strain of Streptomyces chrysogenum producing natamycin: The E. coli ET12567 containing the recombinant plasmid pIB139-epwhiG-dasR obtained in step (5) was transferred into Streptomyces chrysogenum by conjugation transfer method to obtain the genetically engineered strain of Streptomyces chrysogenum producing natamycin.

[0020] The present invention also provides a method for preparing natamycin from the engineered strain of Streptomyces chrysogenum, which involves culturing the engineered strain of Streptomyces chrysogenum in a culture medium at 28–30°C for at least 24 hours.

[0021] In one embodiment, the method includes: first culturing a seed culture in a seed culture medium, and then transferring it to a fermentation culture medium for fermentation to prepare natamycin.

[0022] In one embodiment, the seed culture medium contains yeast powder, glucose, ammonium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, zinc sulfate, and ferrous sulfate.

[0023] In one embodiment, the fermentation medium contains soybean peptone, yeast powder, sodium chloride, magnesium sulfate, and glucose.

[0024] In one embodiment, the method includes the following steps:

[0025] (1) Spore plate culture: Take 100 μL of the strain preserved in the glycerol tube and spread it on MS medium. Incubate at 28℃ for 8-10 days until mature spores grow from a single colony. Then expand the single colony spores to a new MS medium and incubate at 28±0.5℃ for 6-8 days to form a spore plate.

[0026] (2) Seed culture: Two loops of spores were scraped from the MS medium and inoculated into a 250 mL Erlenmeyer flask containing 30 mL of seed culture medium. The flask was then cultured at 28±0.5℃ with shaking at 220 rpm for 24 h.

[0027] (3) Shake flask fermentation culture: 1.8 mL of the seed liquid after 24 h of culture was taken and inoculated into a 250 mL Erlenmeyer flask containing 30 mL of fermentation medium, and cultured at 28±0.5℃ and 220 rpm for 96 h.

[0028] This invention also provides the use of engineered Streptomyces chrysosporium in the preparation of natamycin or products containing natamycin.

[0029] Beneficial effects:

[0030] (1) This invention screened and obtained Streptomyces gilvosporeus strain Z1403, which has improved natamycin production and good passage stability. It can be used as a chassis cell for natamycin production for the construction of genetically engineered bacteria.

[0031] (2) In this invention, epwhiG and dasR were co-expressed in Streptomyces chrysospora Z1403 using transcription factor co-expression technology. The engineered strain obtained achieved a natamycin yield of 2.8 g·L⁻¹ during shake-flask fermentation. -1 The yield was 64.7% higher than that of the starting strain; the yield reached 13.5 g·L⁻¹ in a 5L fermenter. -1 Compared to the original strain (11.2 g·L⁻¹), -1 It increased by 20.5%.

[0032] Preservation of biological materials

[0033] Streptomyces gilvosporeus Z1403, classified as Streptomyces gilvosporeus, was deposited on May 13, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with the deposit number CGMCC No. 34513. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0034] Figure 1 The image shows the screening results of the whiG gene mutant library; where a is the absorbance value of the fermentation broth at 303 nm after fermentation in a 24-well plate of the mutant library; b is the yield and dry weight of the cells after shake-flask fermentation of the mutant library.

[0035] Figure 2 The natamycin yield and cell dry weight of the epwhiG and dasR co-expressed strains were measured.

[0036] Figure 3 A comparison of parameters for 5L fed-batch fermentation of the original strain S. gilvosporeus ATCC 13326 and the high-yield natamycin strain S. gilvosporeus EO-epwhiG-dasR. Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0038] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0039] Unless otherwise specified, the experimental materials used in the following examples are all conventional biochemical reagents.

[0040] (I) Determination of Natamycin Yield

[0041] 1. Determination of natamycin yield by high performance liquid chromatography (HPLC):

[0042] (1) Sample pretreatment: Add 1 mL of fermentation broth to a 15 mL brown centrifuge tube containing 9 mL of 5% glacial acetic acid in methanol. Vortex the mixture for 2 min and centrifuge at 4℃ and 4200 rpm for 20 min. Transfer 4 mL of the supernatant to a 5 mL brown centrifuge tube (determine whether the sample needs dilution based on the preliminary experiment, i.e., the concentration needs to fall within the standard curve), filter with a 1 mL syringe and an organic membrane, and set aside for later use.

[0043] (2) HPLC detection: An Agilent C18 reversed-phase column with a column volume of 250×4.6mm was used. The Agilent 1260 Infinity II mobile phase consisted of 65% methanol and 35% water (ultrapure water was filtered using an aqueous membrane). The column was sonicated for 30 minutes before use to remove air bubbles. The detection wavelength was 303nm, the flow rate was 1mL·min-1, and the injection volume was 10μL.

[0044] 2. Determination of natamycin yield using an enzyme-linked immunosorbent assay (ELISA) reader:

[0045] After fermentation, add 0.5 mL of fermentation broth to a 5 mL brown centrifuge tube containing 4.5 mL of methanol with 5% acetic acid. Vortex the mixture for 2 min and centrifuge at 4℃ and 4200 rpm for 20 min. Transfer 1 mL of the supernatant to a 1.5 mL brown centrifuge tube (to determine if dilution is needed based on preliminary experiments, ensuring the concentration falls within the standard curve). Measure the absorbance at 303 nm using a microplate reader.

[0046] (II) Biomass Measurement Methods

[0047] The growth of *S. gilvosporeus* in shake flasks and 5L fermenters was assessed using cell dry weight (DCW). 4 mL of fermentation broth was centrifuged at 12,000 rpm for 10 min. For the 5L fermenter, 10 mL of fermentation broth was centrifuged at 12,000 rpm for 10 min. The precipitate was filtered onto pre-weighed filter paper and dried in an oven at 105 °C for 12 h before weighing.

[0048] (III) Culture Medium

[0049] MS solid culture medium (g·L) -1 ): Mannitol 20, soybean flour 20, agar 20, pH 7.0, sterilized at 115℃ for 15 min.

[0050] Seed culture medium (g·L) -1 Ingredients: 5g yeast powder, 50g glucose, 10g ammonium sulfate, 1.36g potassium dihydrogen phosphate, 0.8g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.04g zinc sulfate, 0.03g ferrous sulfate, pH 7.0, sterilized at 115℃ for 20min.

[0051] Fermentation medium (g·L) -1 ): Soy peptone 20, yeast powder 4.5, sodium chloride 2, magnesium sulfate 1, glucose 60, pH 7.0, sterilized at 115℃ for 20 min.

[0052] LB solid culture medium (g·L) -1 ): Yeast powder 5, sodium chloride 10, tryptone 10, agar powder 20, pH 7.0, sterilized at 121℃ for 20 min.

[0053] 2×YT medium (g·L) -1 Sterilize at 121°C for 20 minutes with 20g tryptone, 10g yeast extract, 5g sodium chloride, pH 7.0.

[0054] (IV) Main Reagents

[0055] The preparation method of antibiotic stock solution is shown in Table 1.

[0056] Table 1. Preparation of antibiotic stock solutions

[0057]

[0058] After sterilization by filtration through a 0.22 μm filter membrane in a sterile environment, the solution was aliquoted into 1.5 mL EP tubes, 1 mL per tube, and stored at -20°C. Apramycin, kanamycin, chloramphenicol, and nalidixic acid were added to the culture medium at a dosage of 0.1%.

[0059] Preparation of main solutions:

[0060] (1) 10mM MgCl2 solution: Weigh 2.033g magnesium chloride hexahydrate, dissolve it in a small amount of deionized water, then transfer it to a 1000mL volumetric flask, and mix it evenly after making up to volume.

[0061] (2) 10mM CaCl2 solution: Weigh 0.11098g of anhydrous calcium chloride, dissolve it in a small amount of deionized water, then transfer it to a 1000mL volumetric flask, and mix it evenly after making up to volume.

[0062] Example 1: Screening of Streptomyces chrysosporium Z1403

[0063] Using *Streptomyces thuringiensis* ATCC 13326 as the starting strain, mutagenesis was performed according to the method disclosed in the paper "Heavy-ion mutagenesis significantly enhances enduracidin production by Streptomyces fungicidicus". The mutagenized strain and the starting strain were cultured separately in fermentation medium. A strain with a natamycin yield increase of more than 70% and good passage stability was obtained and named *Streptomyces thuringiensis* Z1403. It was then deposited at the China General Microbiological Culture Collection Center.

[0064] Example 2: Selection of high-yield natamycin strains using global transcription machinery engineering

[0065] The genetically engineered *Streptomyces chrysosporium* strain was obtained by performing error-prone PCR on the global transcriptional regulator whiG (nucleotide sequence shown in SEQ ID NO.1), followed by introducing epwhiG into *Streptomyces chrysosporium*. The specific steps are as follows:

[0066] 1. Construction of recombinant plasmid pIB139-epwhiG

[0067] (1) The complete genome sequence of *Streptomyces chrysosporium* was obtained from NCBI. Using the *Streptomyces chrysosporium* genome sequence as a template, upstream and downstream primers targeting the two regulatory genes whiG in the natamycin biosynthesis gene cluster were designed and synthesized. A whiG fragment of approximately 837 bp was amplified from the genomic DNA of strain Z1403. Sequencing analysis confirmed its correctness and consistency with the target fragment. The nucleotide sequence is shown in SEQ ID NO.1. After gel extraction and purification, the fragment was ready for use. Using the amplified whiG as a template, QuickMutation was employed... TM QuickMutation Kit TMRandom mutagenesis was performed using the Random Mutagenesis Kit. After verifying the correctness of the PCR amplification products by 1% agarose gel electrophoresis, the recovered products from error-prone PCRs were digested with EcoRI and NdeI. The purified products were then ligated with plasmid pIB139, which had been digested with EcoRI and NdeI and contained the PermE* promoter, to construct the recombinant plasmid pIB139-epwhiG.

[0068] 2. Preparation of E. coli ET12567 containing recombinant plasmid pIB139-epwhiG

[0069] The pIB139-epwhiG constructed in step 1 was transformed into E. coli DH5α competent cells and plated on a substrate containing 50 μg·mL⁻¹. -1 Incubate overnight on LB agar plates containing apramycin. Randomly select colonies for colony PCR verification; the positive transformation rate must be greater than 95%. Wash off the transformants obtained from E. coli DH5α with sterile water and transfer to a plate containing 50 μg / mL apramycin. -1 The cells were cultured overnight in LB broth containing apramycin, and the recombinant expression vector pIB139-epwhiG was extracted and stored at -20°C. The extracted high-concentration pIB139-epwhiG was transformed into E. coli ET12567 competent cells, and the cultured bacterial suspension was plated onto fresh solid LB agar plates containing apramycin, kanamycin, and chloramphenicol resistance. The plates were incubated at 37°C for 16-18 hours. After transformants appeared, colony PCR was performed for verification; the positive transformation rate should be greater than 95%. All transformants were inoculated into 50 mL of LB broth containing kanamycin, chloramphenicol, and apramycin, and cultured at 37°C and 200 rpm until OD500. 600 =0.35-0.6. Collect 35 mL of bacterial culture into a 50 mL centrifuge tube, centrifuge at 8000 rpm for 5 min at 4 °C, and discard the supernatant under aseptic conditions. Then wash the bacterial precipitate with 10 mL of LB liquid medium, repeat 2-3 times, and then suspend the bacterial cells in 3 mL of LB medium for later use.

[0070] 3. Construction of the whiG gene mutation library

[0071] (1) Using an inoculation spatula, scrape the spores from the prepared Streptomyces chrysosporium Z1403 spore plate into a shake flask containing 20 mL of sterile water and glass beads. After dispersing the spores, filter them into a sterile empty bottle using a syringe containing gauze. Add 20 mL of 2×YT medium and heat-shock in a 50°C water bath for 10 min. Germinate in a shaker at 28°C and 220 rpm for 2.5 h. After germination, centrifuge at 4°C and 8000 rpm for 5 min. Under sterile conditions, discard the supernatant and add 3 mL of LB medium to suspend the spore solution for later use.

[0072] (2) Mix the pretreated E. coli ET12567 and Streptomyces chrysogenum Z1403 spore suspensions containing pIB-epwhiG from step (1), centrifuge at 8000 rpm for 5 min at 4°C, discard the supernatant, and resuspend in 600 μL of LB medium. Spread 100 μL of the bacterial suspension onto MS agar plates containing magnesium chloride, incubate at 28°C for 24 h, and then cover with apramycin and nalidixic acid. Afterward, incubate at 28°C for 5-7 days. Once conjugates have grown, transfer them to MS agar plates containing apramycin resistance using sterile toothpicks for plating verification.

[0073] 4. Preparation of natamycin using strains

[0074] (1) 24-well plate culture: Mature single spores were inoculated into 24-well plates containing 2 mL of seed culture using an inoculation loop and cultured at 28 °C and 220 rpm for 24 h. Then, the seed culture was transferred to 24-well plates containing 2 mL of fermentation medium at an inoculation rate of 6% and fermented at 28 °C and 220 rpm for 72 h.

[0075] (2) Detection of natamycin concentration using an ELISA reader: After fermentation, take 0.5 mL of the fermentation broth prepared in step (1) and add it to a 5 mL brown centrifuge tube containing 4.5 mL of 5% acetic acid and methanol. Vortex the mixture for 2 min and centrifuge at 4℃ and 4200 rpm for 20 min. Take 1 mL of the supernatant into a 1.5 mL brown centrifuge tube (based on preliminary experiments, determine whether the sample needs dilution, i.e., the concentration needs to fall within the standard curve). Measure its absorbance at 303 nm using an ELISA reader. The results are as follows: Figure 1 (a).

[0076] (3) Shake-flask culture: The top five strains with the highest absorbance values ​​were inoculated into seed culture medium and cultured at 28℃ and 220 rpm for 24 h to obtain seed culture. The seed culture was then inoculated into fermentation medium at a 6% inoculation rate and cultured at 28℃ and 220 rpm for 96 h. The natamycin yield was then determined. Figure 1(b) A total of 5 strains were screened, among which strain EP-2 had the highest yield, with a shake flask yield of 2.3 g·L⁻¹. -1 .

[0077] Example 3: Construction of a high-yield natamycin strain through global transcription factor co-expression

[0078] 1. Construction of recombinant plasmid pIB-dasR

[0079] The complete genome sequence of *Streptomyces chrysosporium* was obtained from NCBI. Using the *Streptomyces chrysosporium* genome sequence as a template, upstream and downstream primers targeting the regulatory gene dasR in the natamycin biosynthesis gene cluster were designed and synthesized. A fragment of dasR (SEQ ID NO. 3) was amplified from the genomic DNA of strain Z1403 obtained in Example 1, and purified by gel extraction for later use. The purified product was digested with EcoRI and NdeI. The purified product was ligated with the plasmid pIB139 containing the PermE* promoter, which had already been digested with EcoRI and NdeI, to construct the recombinant plasmid pIB139-dasR.

[0080] 2. Construction of recombinant plasmid pIB139-epwhiG-dasR

[0081] Using plasmid PAcas9 as a template, the T0 fragment (nucleotide sequence shown in SEQ ID NO. 4) was amplified using T0-F and T0-R primers, with a fragment size of 391 bp. Using the genome of strain EP-2 obtained in Example 1 as a template, the Perm-epwhiG fragment containing the promoter PermE* (nucleotide sequence shown in SEQ ID NO. 5) was amplified using Perm-epwhiG-F and Perm-epwhiG-R primers, with a fragment size of 1038 bp. The successfully constructed plasmid pIB139-dasR was used as a vector, digested with EcoRI, and ligated to the aforementioned constructed T0 fragment and Perm-epwhiG. After ligation, the cells were transformed into E. coli DH5α. After transformants grew, colony PCR was performed using the verification primers M13-F-47 and M13-R-48. If the ligation was correct, a fragment of 2445 bp could be amplified. The PCR amplification product was verified by 1% agarose gel electrophoresis.

[0082] 3. Preparation of E. coli ET12567 containing recombinant plasmid pIB139-epwhiG-dasR

[0083] The ligation product prepared in step 1 was transformed into E. coli DH5α competent cells and coated onto a substrate containing 50 μg·mL⁻¹. -1The cells were cultured overnight on LB agar plates containing apramycin, and colonies were randomly selected for colony PCR verification. After successful verification, the colonies were inoculated into 5 mL of LB liquid medium and cultured overnight. The recombinant plasmid was then extracted and sent to a third-party company for sequencing and sequence alignment. The verified recombinant plasmid pIB139-epwhiG-dasR was extracted. The extracted recombinant plasmid pIB139-epwhiG-dasR was transformed into E. coli ET12567 competent cells, and the cultured bacterial solution was plated on fresh LB agar plates containing apramycin, kanamycin, and chloramphenicol resistance. The plates were incubated at 37°C for 16-18 hours. After transformants appeared, colony PCR was performed for verification. The correctly verified transformants were inoculated into 5 mL of LB liquid medium containing kanamycin, chloramphenicol, and apramycin, and incubated overnight at 37°C and 200 rpm. Then, they were transferred to 50 mL of fresh LB liquid medium containing kanamycin, chloramphenicol, and apramycin, and incubated at 37°C and 200 rpm until OD (outlet count) was reached. 600 =0.35-0.6. Collect 35 mL of bacterial culture into a 50 mL centrifuge tube, centrifuge at 8000 rpm for 5 min at 4 °C, and discard the supernatant under aseptic conditions. Then wash the bacterial precipitate with 10 mL of LB liquid medium, repeat 2-3 times, and then suspend the bacterial cells in 3 mL of LB medium for later use.

[0084] 4. Preparation of genetically engineered *Streptomyces chrysosporium* strains for natamycin production

[0085] (1) Using an inoculation spatula, scrape the spores from the prepared Streptomyces chrysosporium Z1403 spore plate into a shake flask containing 20 mL of sterile water and glass beads. After dispersing the spores, filter them into a sterile empty bottle using a syringe containing gauze. Add 20 mL of 2×YT and heat-shock in a 50°C water bath for 10 min. Germinate in a shaker at 28°C and 220 rpm for 2.5 h. After germination, centrifuge at 4°C and 8000 rpm for 5 min. Under sterile conditions, discard the supernatant and add 3 mL of LB medium to suspend the spore solution for later use.

[0086] (2) The pretreated spore suspensions of *E. coli* ET12567 and *Streptomyces chrysospora* Z1403 containing the pIB139-epwhiG-dasR plasmid were mixed and centrifuged at 8000 rpm for 5 min at 4 °C. The supernatant was discarded, and the suspension was resuspended in 600 μL of LB medium. 100 μL of the bacterial suspension was spread on MS agar plates containing magnesium chloride and incubated at 28 °C for 24 h. Then, apramycin and nalidixic acid were added. The plates were then incubated at 28 °C for 5-7 days. After conjugates grew, they were transferred to MS agar plates containing apramycin resistance using sterile toothpicks for plastisol spotting verification.

[0087] 5. Preparation of natamycin using strains

[0088] (1) Seed culture: The top five strains with the largest absorbance values ​​were inoculated into seed culture medium and cultured in a shaker at 28℃ and 220rpm for 24h to obtain seed culture.

[0089] (2) Fermentation medium culture: The seed culture obtained in step (1) was inoculated into the fermentation medium at an inoculum size of 6%, and cultured for 96 h in a shaker at 28℃ and 220 rpm. The results are as follows: Figure 2 As shown, the strain with the highest yield was named EO-epwhiG-dasR.

[0090] like Figure 2 As shown, natamycin production was gradually increased through transcriptional machinery engineering and co-expression of transcription factors. The obtained engineered strain achieved a yield of 2.8 g·L⁻¹ in shake-flask fermentation. -1 This represents a 64.7% increase compared to the original strain.

[0091] Example 4: Natamycin prepared using a fed-batch method from *Streptomyces chrysogenum* EO-epwhiG-dasR, a high-yield natamycin spore-producing strain.

[0092] Preparation of seed culture: The starting strain Z1403 and the high-yielding strain EO-epwhiG-dasR, which were streaked on MS plates, were inoculated into 30 mL of sterile seed culture medium. The medium was placed on a shaker and cultured at 28 °C and 200 rpm for 30 h to obtain the seed culture of Z1403 and EO-epwhiG-dasR.

[0093] Fermentation culture: 240 mL of pre-cultured Z1403 and EO-epwhiG-dasR seed culture was inoculated into 3.26 L of sterile fermentation medium, with an initial pH of 7.0. The pH was maintained at 6.0 ± 0.1 by adding 12.5% ​​NH4OH, and dissolved oxygen was maintained at 30% by adjusting the aeration speed and aeration rate. When the glucose concentration was below 20 g·L⁻¹... -1 At that time, sterilized glucose (50%) is automatically added to maintain its concentration at approximately 20 g / L. -1 Fermentation was completed in 120 hours, and fermentation broth samples were collected every 12 hours for analysis during the fermentation process.

[0094] The yield and cell count of natamycin in the fermentation broth were determined, such as... Figure 3 As shown, the two strains exhibited similar trends in natamycin production. Natamycin accumulated rapidly between 0 and 70 hours, reaching its peak at 120 hours, with strain Z1403 achieving a natamycin yield of 11.3 g·L⁻¹. -1 At this point, the yield of EO-epwhiG-dasR natamycin reached 13.5 g·L⁻¹.-1 Regarding cell count, the cell count of Z1403 increased rapidly in the early stage, and basically entered a stable period after the third day, with a final cell dry weight of 34.3 g·L. -1 Comparative Example 1:

[0095] The specific implementation method is the same as in Example 2, except that a recombinant plasmid pIB139-whiG was constructed and transformed into *Streptomyces chrysosporium* Z1403. Shake-flask fermentation was then performed according to the method in Example 2. The results showed that the natamycin yield was 2.0 g·L⁻¹ after 96 h of fermentation. -1 The yield was increased by 15% compared to the starting strain.

[0096] Comparative Example 2:

[0097] The specific implementation method is the same as in Example 2, except that the recombinant plasmid pIB139-epwhiG was transformed into *Streptomyces chrysosporium* Z1403, and shake-flask fermentation was carried out according to the method in Example 2. The results showed that the yield of natamycin after 96 hours of fermentation was 2.3 g·L⁻¹. -1 The yield increased by 35% compared to the starting strain.

[0098] Comparative Example 3:

[0099] The specific implementation method is the same as in Example 2, except that a recombinant plasmid pIB139-dasR was constructed and transformed into *Streptomyces chrysosporium* Z1403. Shake-flask fermentation was then performed according to the method in Example 2. The results showed that the yield of natamycin after 96 hours of fermentation was 2.4 g·L⁻¹. -1 The yield increased by 41% compared to the starting strain.

[0100] 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 determined by the claims.

Claims

1. An engineered strain of *Streptomyces chrysosporium*, characterized in that, The epwhiG gene and the global transcription factor dasR were expressed; the nucleotide sequence of the epwhiG gene is shown in SEQ ID NO.2; the nucleotide sequence of the dasR gene is shown in SEQ ID NO.

3.

2. The engineered *Streptomyces chrysosporium* strain according to claim 1, characterized in that, The expression of the epwhiG gene and the global transcription factor dasR is regulated using the promoter shown in SEQ ID NO.

6.

3. The engineered *Streptomyces chrysosporium* strain according to claim 1, characterized in that, The engineered strain of Streptomyces chrysosporium is Streptomyces chrysosporium Z1403 as the starting strain; Streptomyces chrysosporium Z1403 was deposited at the China General Microbiological Culture Collection Center on May 13, 2025, with the preservation number CGMCC No. 34513.

4. The application of the epwhiG gene in increasing the natamycin yield of engineered Streptomyces chrysogenum, characterized in that, The nucleotide sequence of the epwhiG gene is shown in SEQ ID NO.

2.

5. A method for increasing the yield of natamycin from *Streptomyces thuringiensis*, characterized in that, Includes the following steps: (1) Insert the dasR gene, T0 fragment, and Perm-epwhiG fragment into the multiple cloning site of the pIB139 vector plasmid to obtain the recombinant plasmid pIB139-epwhiG-dasR. (2) Transform the recombinant plasmid pIB139-epwhiG-dasR constructed in step (1) into E. coli ET12567; (3) The transformant from step (2) was transferred into Streptomyces chrysogenum via a binding transfer method to obtain an engineered Streptomyces chrysogenum strain that produces natamycin.

6. Streptomyces gilvosporeus Z1403 was deposited on May 13, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with the deposit number CGMCC No. 34513.

7. A method for preparing natamycin, characterized in that, The engineered strain of Streptomyces chrysogenum according to any one of claims 1 to 3 or Streptomyces chrysogenum Z1403 according to claim 6 is cultured in a culture medium at 28 to 30°C for at least 24 hours.

8. The method according to claim 7, characterized in that, The method includes the following steps: (1) Spore plate culture: The engineered strain of Streptomyces chrysogenum was cultured in MS medium at 28±0.5℃ to form spores; (2) Seed culture: The spores prepared in step (1) are inoculated into the seed culture medium and cultured at 28±0.5℃ for at least 24h; (3) Fermentation culture: The seed liquid prepared in step (2) is cultured in a fermentation medium at 28±0.5℃ for at least 96h.

9. The method according to claim 8, characterized in that, The seed culture medium contains yeast extract, glucose, ammonium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, zinc sulfate, and ferrous sulfate; and / or The fermentation medium contains soybean peptone, yeast powder, sodium chloride, magnesium sulfate, and glucose.

10. The engineered strain of Streptomyces chrysogenum according to any one of claims 1 to 3, or the Streptomyces chrysogenum Z1403 according to claim 6, in the preparation of natamycin or products containing natamycin.

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