Streptomyces engineering bacteria for high yield of natamycin and construction method and application thereof

Abstract

The invention discloses a streptomyces engineering bacterium for high yield of natamycin and a construction method thereof. According to the invention, an original strain is subjected to electron accelerator mutagenesis combined with gene engineering, such that a streptomyces engineering strain with high natamycin yield is successfully constructed. The engineering strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on July 28, 2025, the classification name of the engineering strain is Streptomyces gilvosporeus QZ103, and the preservation number of the engineering strain is CGMCC No.35422. The invention also discloses a preparation method of the engineering strain. The yield of natamycin produced by fermenting the engineering strain provided by the invention in a shake flask reaches 6.4 g / L, which is 8.42 times that of an original strain; the fermentation yield in a 15L fermentation tank reaches 29.2 g / L, and the strain has good passage stability and is suitable for large-scale production requirements of natamycin.

Description

Technical Field

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

[0002] The molecular formula of natamycin is C 33 H 47 NO 13 Natamycin is a polyene macrolide antifungal antibiotic with a 26-carbon skeleton. It is a white or off-white crystalline powder. Originally isolated from *Streptomyces natalatum*, it is also produced by several other *Streptomyces* species, including *Streptomyces chatanuga*, *Streptomyces flavus*, and *Streptomyces lidi*. Natamycin exhibits strong broad-spectrum antifungal activity even at very low concentrations and is the only antifungal drug recognized as GRAS (Generally Recognized As Safe) by the US FDA. For nearly 50 years, it has been widely used as a natural preservative and antifungal agent in food, medicine, agriculture, and other fields. According to statistics, the global production value of natamycin was approximately US$190 million in 2023 and is projected to reach US$290 million by 2030. With the continuous development of natamycin in pharmaceuticals and food, market demand continues to grow. However, the current low fermentation level and high production cost of natamycin limit its further development and application. Therefore, obtaining superior high-yield strains to reduce production costs has become a current research hotspot.

[0003] The main methods for obtaining high-yielding natamycin strains include mutagenesis and genetic engineering. In current industrial production, traditional mutagenesis screening methods still dominate, but these methods are time-consuming and labor-intensive. Continuous use of traditional mutagenesis techniques easily encounters bottlenecks, failing to achieve sustained improvements in strain performance and even leading to a significant decrease in strain stability. Summary of the Invention

[0004] The purpose of this invention is to address the problem of low natamycin production by providing a high-yield *Streptomyces* strain, its construction method, and its applications. This invention utilizes a combination of electron accelerator mutagenesis and genetic engineering techniques to modify the starting strain, thereby increasing natamycin synthesis efficiency and yield.

[0005] The technical solution adopted in this invention is:

[0006] I. A Streptomyces strain that produces high levels of natamycin

[0007] The Streptomyces species mentioned are named Streptomyces gilvosporeus QZ101, with accession number CGMCC No.35421, and the depository institution is the China General Microbiological Culture Collection Center.

[0008] II. A high-natamycin-producing engineered Streptomyces strain

[0009] The engineered Streptomyces strain was obtained by mutagenesis of Streptomyces gilvosporeus QZ101 using electron accelerator mutagenesis technology, followed by the introduction of a BAC plasmid containing a natamycin synthesis gene cluster through genetic engineering.

[0010] Preferably, the natamycin synthesis gene cluster is derived from Streptomyces chatanuga.

[0011] Preferably, the natamycin synthesis gene cluster is derived from Streptomyces chattanoogensis L10, NCBI number HQ386234.1.

[0012] Preferably, the engineered Streptomyces strain is named Streptomyces gilvosporeus QZ103, with accession number CGMCC No. 35422, and the depository is the China General Microbiological Culture Collection Center.

[0013] III. A method for constructing a high-natamycin-producing engineered Streptomyces strain

[0014] The construction method includes the following steps:

[0015] S1. The starting strain is subjected to at least one round of irradiation mutagenesis treatment using an electron accelerator to obtain a mutant strain; the starting strain is the Streptomyces strain as described in claim 1; the treatment dose during the irradiation mutagenesis treatment is 1-5 kGy.

[0016] Specifically, in each round of irradiation mutagenesis treatment, multiple dose groups with increasing gradients were selected in the range of 1 to 5 kGY to irradiate the spore suspension. After irradiation and culture, multiple single colonies were randomly selected from each dose group for screening.

[0017] S2. The BAC plasmid containing the natamycin synthesis gene cluster was introduced into the mutant strain to obtain the engineered Streptomyces strain.

[0018] IV. Application of one of the above-mentioned Streptomyces strains or the above-mentioned engineered Streptomyces strains in the fermentation production of natamycin.

[0019] V. A method for producing natamycin by fermentation using the above-mentioned Streptomyces strain or the above-mentioned engineered Streptomyces strain.

[0020] The method for producing natamycin by fermentation includes the following steps:

[0021] D1. Inoculate the strain into seed culture medium and culture it in a constant temperature shaker at a temperature of 28-32℃ until the cell volume reaches 4-6%, thus obtaining the seed liquid.

[0022] Specifically, the components and their mass-volume ratios contained in the seed culture medium are as follows: glucose content of 1-3%, peptone content of 1-3%, NaCl content of 0.25-1%, and pH of 6.0-7.5.

[0023] S2. The seed liquid is inoculated into the fermentation medium at a volume inoculation rate of 3-5%, and fermented at a temperature of 28-32℃ for 120-168 hours to obtain a fermentation broth containing natamycin.

[0024] Specifically, the fermentation medium contains the following components and their mass-volume ratios: glucose content of 4-8%, peptone content of 0.5-3%, yeast powder content of 0.5-3%, magnesium sulfate heptahydrate content of 0.05-0.3%, calcium carbonate content of 0.05-0.4%, and pH of 6.0-7.5; glucose in the fermentation medium is added after being sterilized separately.

[0025] The beneficial effects of this invention are:

[0026] 1. This invention is the first to propose the application of electron accelerator mutagenesis technology to the mutagenesis method of natamycin-producing bacteria, and the method has a higher mutation rate and a more significant yield improvement effect.

[0027] 2. This invention uses a combination of mutagenesis and genetic engineering techniques to efficiently obtain a high-yield streptomycin-producing strain QZ103. The strain achieves a yield of 6.4 g / L in shake-flask fermentation, which is 8.42 times that of the original strain. In a 15L fermenter, the yield reaches 29.2 g / L, which is 269% higher than that of the original strain.

[0028] 3. The method of this invention effectively improves the fermentation performance of the strain through genetic engineering technology, and when combined with mutagenesis technology, it can efficiently improve the performance of the strain. Attached Figure Description

[0029] Figure 1 This is a colony morphology diagram of strain QZ101.

[0030] Figure 2 The image shows the shake-flask fermentation results of the starting strain QZ101 and the mutant strain QZ102-3 in this invention.

[0031] Figure 3 This is a schematic diagram of the structure of the BAC plasmid pMSBBAC1-NT in this invention.

[0032] Figure 4 The image shows the shake-flask fermentation results of the starting strain QZ101, the mutant strain QZ102-3, and the engineered strain QZ103 in this invention.

[0033] Figure 5 This is a graph showing the trend of natamycin production in a 15L fermenter for the starting strain QZ101 and the engineered strain QZ103 in this invention. Detailed Implementation

[0034] 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.

[0035] This invention provides a Streptomyces strain that produces high levels of natamycin. The Streptomyces strain is named *Streptomyces gilvosporeus* QZ101, with accession number CGMCC No. 35421. It is deposited at the China General Microbiological Culture Collection Center on July 28, 2025, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0036] This invention also provides a high-yield natamycin-producing engineered Streptomyces strain. The engineered Streptomyces strain was obtained by mutagenesis and screening of Streptomyces gilvosporeus QZ101 using electron accelerator mutagenesis technology, followed by the introduction of a BAC plasmid containing a natamycin synthesis gene cluster through genetic engineering.

[0037] Preferably, the natamycin synthesis gene cluster is derived from Streptomyces chatanuga.

[0038] Preferably, the natamycin synthesis gene cluster is derived from Streptomyces chattanoogensis L10.

[0039] Preferably, the engineered Streptomyces strain is named Streptomyces gilvosporeus QZ103, with accession number CGMCC No.35422, deposited at the China General Microbiological Culture Collection Center, on July 28, 2025, and located at Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0040] The present invention also provides a method for constructing a Streptomyces strain that produces high levels of natamycin.

[0041] Specifically, the steps include the following:

[0042] S1. At least one round of irradiation mutagenesis treatment was carried out on Streptomyces chrysosporium QZ101 using an electron accelerator, and mutant strains were obtained after screening.

[0043] Preferably, the treatment dose during the irradiation mutagenesis process is 1–5 kGy.

[0044] Preferably, in each round of irradiation mutagenesis treatment, multiple dose groups with increasing gradients are selected in the range of 1 to 5 kGY to irradiate the spore suspension. After irradiation and culture, multiple single colonies are randomly selected from each dose group for screening to obtain mutant strains.

[0045] Preferably, the screening process is as follows: First, 50 single colonies from each dosage group are randomly selected and cultured on YMG solid medium, and initial screening is performed using the agar column method. Subsequently, the concentration of natamycin in the fermentation broth is detected by HPLC, and finally, mutant strains are obtained.

[0046] S2. The BAC plasmid containing the natamycin synthesis gene cluster was introduced into the mutant strain to obtain the engineered Streptomyces strain.

[0047] Preferably, a BAC plasmid containing a natamycin synthesis gene cluster is introduced via a three-parental conjugation method.

[0048] In a specific embodiment of the present invention, the initial vector for the BAC plasmid pMSBBAC1-NT containing the natamycin synthesis gene cluster is the pMSBBAC1 plasmid.

[0049] The present invention also provides the application of the above-mentioned Streptomyces strain or the above-mentioned engineered Streptomyces strain in the fermentation production of natamycin.

[0050] The present invention also provides a method for producing natamycin by fermentation using the above-mentioned Streptomyces strain or the above-mentioned engineered Streptomyces strain.

[0051] Specifically, the steps include the following:

[0052] D1. Inoculate the strain into seed culture medium and culture it in a constant temperature shaker at a temperature of 28-32℃ and a rotation speed of 180-230rpm for 16-24h until the bacterial cell volume reaches 4-6%, thus obtaining the seed liquid.

[0053] Preferably, the culture is carried out in a constant temperature shaker at a temperature of 30°C and a rotation speed of 230 rpm for 16–24 hours.

[0054] S2. Inoculate the seed culture into the fermentation medium at a volume of 3-5%, and ferment at a temperature of 28-32℃ and a rotation speed of 180-230 rpm for 120-168 h to obtain a fermentation broth containing natamycin.

[0055] Preferably, the seed liquid is inoculated into the fermentation medium at a volume concentration of 5%, and fermented at a temperature of 30°C and a rotation speed of 230 rpm for 120 h to obtain a fermentation broth containing natamycin.

[0056] Preferably, the components and their mass-volume ratios in the seed culture medium are as follows: glucose content 1-3%, peptone content 1-3%, NaCl content 0.25-1%, pH 6.0-7.5, sterilized at 115-121℃ for 15-30 min.

[0057] Most preferably, the seed culture medium contains the following components and their mass-volume ratios: 2% glucose, 1.5% peptone, 1% NaCl, pH 7.0, and is sterilized at 115°C for 30 min.

[0058] Preferably, the fermentation medium contains the following components and their mass-volume ratios: glucose content of 4-8%, peptone content of 0.5-3%, yeast powder content of 0.5-3%, magnesium sulfate heptahydrate content of 0.05-0.3%, calcium carbonate content of 0.05-0.4%, pH of 6.0-7.5, and is sterilized at 115-121℃ for 15-30 min; glucose in the fermentation medium is added after being sterilized separately.

[0059] Most preferably, the fermentation medium contains the following components and their mass-volume ratios: glucose content of 4-8%, peptone content of 2.5%, yeast powder content of 1%, magnesium sulfate heptahydrate content of 0.02%, calcium carbonate content of 0.03%, pH of 7.0, and sterilized at 115°C for 30 min; glucose in the fermentation medium is added after being sterilized separately.

[0060] Specific embodiments of the present invention are as follows:

[0061] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0062] Unless otherwise specified, the experimental materials used in the following examples are all conventional biochemical reagents.

[0063] (I) Determination of Natamycin by HPLC

[0064] HPLC detection conditions:

[0065] Chromatographic column: Welchrom C18, 5µm, 4.6mm × 250mm; Mobile phase: Weigh 3.0g ammonium acetate and 1.0g ammonium chloride, dissolve in 760mL water, and mix well. Add 5.0mL tetrahydrofuran and 240mL acetonitrile, mix well, and filter through a 0.45μm membrane filter; Flow rate: 1.0mL / min; Detection wavelength: 303nm; Injection volume: 10µl; Column temperature: 30℃.

[0066] Sample processing:

[0067] Take 1 mL of fermentation broth, add 9 mL of methanol and mix well. Sonicate for 30 min and centrifuge at 8000 rpm for 10 min. Dilute the supernatant with methanol to an appropriate factor and filter through a 0.22 μm organic filter membrane to obtain the sample to be tested.

[0068] (II) Determination of PMV (Particle Volume)

[0069] Take 5 mL of seed culture medium into a 10 mL centrifuge tube and centrifuge at 4000 rpm for 10 min. Measure the volume of the supernatant.

[0070]

[0071] (III) Culture Medium

[0072] MS solid medium: mannitol 2%, soybean powder 2%, agar powder 2%;

[0073] YMG solid medium: yeast extract 0.4%, malt extract 1%, glucose 0.4%, agar powder 1.5-2%, calcium carbonate 0.2%, pH 7.0;

[0074] TSB medium: 2% TSB powder (BD Difco), 50% PEG6000 flakes;

[0075] ISP2 medium: yeast extract 0.4%, malt extract 1%, glucose 0.4%, agar 2%;

[0076] ISP4 medium: 3.7% (BD ISP4 Medium), 20 mM magnesium chloride, 50 mM calcium chloride;

[0077] Seed culture medium: 2% glucose, 1.5% peptone, 1% sodium chloride, pH 7.0;

[0078] Fermentation medium: glucose 4-8%, peptone 2%, yeast extract 1%, pH 7.0;

[0079] All the above culture medium components are mass-volume ratios.

[0080] Example 1

[0081] This embodiment demonstrates the isolation and identification of a natamycin-producing Streptomyces strain. Sample source: isolated from soil. The specific process is as follows:

[0082] (1) Soil sample collection

[0083] Soil samples were collected from a soil sample collection site (28°35′N 121°24′E). Soil samples were dug from different locations at a depth of 8–15 cm using a spatula and placed in sterile sampling tubes for preservation. A total of 100 samples were collected. The sampling tubes were brought back to the laboratory, and the soil samples were left to stand in a ventilated environment for seven days to allow moisture to evaporate.

[0084] (2) Preparation of soil suspension

[0085] Weigh out a small clump of soil from a 5g soil sample, dry it, and grind it into powder. Pour the powder into a 250mL Erlenmeyer flask containing 30mL of sterile water and small glass beads, and sonicate for 30 minutes to obtain the stock solution. Use a pipette to transfer 100μL of the soil stock solution into an EP tube containing 900μL of sterile water, and mix thoroughly to obtain a 10-fold diluted soil suspension. Repeat the dilution process as described above to obtain soil suspensions with dilution factors of 10. 2 10 3 10 4 10 5 10 6 The suspension was diluted five times, and Streptomyces was isolated on the isolation medium using these five concentrations of dilution.

[0086] (3) Isolation of actinomycetes

[0087] ISP4 solid medium was selected as the isolation medium, dispensed in 100 mL portions, and sterilized at 121°C for 30 min. After sterilization, the medium was cooled to approximately 80°C, and nalidixic acid was added to a final concentration of 25 μg / mL to inhibit the growth of Gram-negative bacteria, and nystatin was added to a final concentration of 50 μg / mL to inhibit fungal growth. Among the colonies that grew, Streptomyces samples with spore-producing colonies were selected for preservation, resulting in the collection of 70 strains.

[0088] (4) Fermentation of actinomycetes

[0089] Select strains with good colony morphology (e.g., size / wrinkles / color), streak them onto ISP4 solid medium, and incubate at 28°C for 7 days. Scrape a single colony and inoculate it into a 250mL Erlenmeyer flask containing 30mL of seed medium, and incubate at 28°C and 230rpm for 24 hours. Once the mycelium has matured and no contamination is observed under a microscope, transfer 3mL of bacterial culture from the seed medium to a 250mL Erlenmeyer flask containing 30mL of fermentation medium. Set up three replicates for each strain, and incubate at 28°C and 230rpm for 5 days. Detect the concentration of natamycin in the fermentation broth using HPLC. Based on the HPLC results, the strain with the highest yield, QZ101, was cultured on YMG solid medium at 28°C for approximately 7 days, and the cells were preserved in 20% glycerol at -80°C.

[0090] (5) Strain identification

[0091] Morphological observation: strain QZ101 was cultured on ISP4 medium plates at 28°C for 5–7 days. Figure 1 The image shows the colony morphology of strain QZ101. The colonies are grayish-white, with good growth of both intracellular and aerial hyphae. The phenotypic characteristics in various culture media are shown in Table 1.

[0092] Table 1. Epigenetic characteristics of the strains

[0093]

[0094] Example 2

[0095] This embodiment demonstrates the process of screening high-yield mutant strains using electron accelerator mutagenesis.

[0096] The specific process is as follows:

[0097] (1) Using *Streptomyces chrysosporium* QZ101, screened in Example 1 above, as the starting strain, it was streaked on YMG solid medium and cultured at 30°C for 5–7 days. Spores were collected with 20% glycerol solution and prepared into a spore suspension, which was then irradiated using an electron accelerator. The electron accelerator was provided by the Institute of Nuclear Agricultural Sciences, Zhejiang University. The spore suspension was irradiated in five dose groups: 1 kGY, 2 kGY, 3 kGY, 4 kGY, and 5 kGY. Immediately after irradiation, the suspension was diluted and spread onto MS solid medium and cultured at 30°C for 5–7 days. Fifty single colonies from each dose group were randomly selected and expanded on YMG solid medium, and initial screening was performed using the agar column method. The indicator strain was *Saccharomyces cerevisiae*. The detection medium consisted of YPD medium with 20 g / L agar powder at the bottom and YPD medium with 15 g / L agar powder at the top, containing a certain amount of indicator strain. Ten mutant strains with the largest inhibition zone diameter were selected from each dosage group, totaling 50 strains. These strains were inoculated into seed culture medium and cultured at 30℃ and 230 rpm for 18–24 h. Then, they were inoculated into fermentation medium at a volume fraction of 3% and cultured at 30℃ and 230 rpm for 120 h. The concentration of natamycin in the fermentation broth was detected by HPLC, ultimately yielding the high-yielding mutant strain QZ102-1 from the first round of mutagenesis.

[0098] In practice, the performance of strains treated with different intensities of mutagenesis will change to varying degrees, with the improvement effect exhibiting a parabolic change as the intensity increases. In this embodiment, high-yielding mutant strains were screened out at 2kGY and 3kGY levels.

[0099] (2) Using QZ102-1 as the starting strain obtained in step (1), a spore suspension was prepared and subjected to electron accelerator mutagenesis as described in step (1). The suspension was then spread onto MS solid medium and incubated at 30°C for 5–7 days. Fifty single colonies from each dose group were randomly selected and expanded on YMG solid medium, and initial screening was performed using the agar column method. The concentration of natamycin in the fermentation broth was detected by HPLC, and the mutant strain QZ102-2, which produced high yields in the second round of mutagenesis, was finally obtained.

[0100] (3) Using the strain QZ102-2 obtained in step (2) as the starting strain, a spore suspension was prepared. Electron accelerator mutagenesis was performed according to the method in step (1), and the suspension was spread onto MS solid medium and cultured at 30℃ for 5–7 days. Fifty single colonies from each dosage group were randomly selected and expanded on YMG solid medium, and initial screening was performed using the agar column method. The concentration of natamycin in the fermentation broth was detected by HPLC, and the high-yielding mutant strain QZ102-3 from the third round of mutagenesis was finally obtained. The shake-flask fermentation results are as follows: Figure 2 As shown, the yield of QZ102-3 reached 3.51 g / L, which was 361% higher than that of the original strain.

[0101] Example 3

[0102] This embodiment constructs a high-natamycin-producing Streptomyces engineered strain. Specifically, in this embodiment, the BAC plasmid pMSBBAC1-NT containing the natamycin synthesis gene cluster provided by the Institute of Pharmaceutical Biotechnology of Zhejiang University was introduced into the mutant strain QZ102-3 through triparental conjugation to obtain the high-natamycin-producing engineered strain QZ103.

[0103] The construction process is as follows:

[0104] (1) A schematic diagram of the structure of plasmid pMSBBAC1-NT is shown below. Figure 3 As shown, pMSBBAC1-NT carries the following main elements: a natamycin synthesis gene cluster from *Streptomyces chatanuga* (NCBI number HQ386234.1), the aac(3)Ⅳ resistance gene, and the ΦC31 integration site, and is stored in *Escherichia coli* DH10B. The primers listed in Table 2 were used to verify whether the BAC plasmid contains the natamycin synthesis gene cluster. The *E. coli* strain was inoculated into 5 mL of LB medium containing 50 μg / mL apramycin and cultured overnight at 37°C. *E. coli* ET12567 / pUB307 was inoculated into 5 mL of LB medium containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol and cultured overnight at 37°C and 220 rpm.

[0105] Primers used (Table 2)

[0106]

[0107] (2) Take 0.2 mL of the above bacterial culture solution and transfer it to 10 mL of LB liquid medium containing the corresponding antibiotic. Incubate at 37°C and 220 rpm for 2–3 h until OD500 is reached. 600 The volume was approximately 0.4–0.6. All bacterial cells were collected by centrifugation at 4000 rpm. The bacterial cells were washed twice with fresh LB medium and then resuspended in 0.5 mL of LB medium.

[0108] (3) Mix the two types of E. coli from step (2) at a 1:1 ratio, spread them on LB solid medium, and incubate at 37°C for 22–24 h. Streak the bacterial colony onto LB solid medium containing three antibiotics (amprolium 50 μg / mL, kanamycin 50 μg / mL, and chloramphenicol 25 μg / mL), and incubate upside down at 37°C for 24 h until single colonies emerge. Identify positive single colonies by colony PCR. Use single colonies as templates. Perform PCR verification using pMS-F / pMS-R (Table 3) as primers. The 20 μL PCR reaction system contains the following components: 2×Phanta Max Master Mix, 10 μL; pMS-F, 1 μL; pMS-R, 1 μL; colony dilution buffer, 0.5 μL; sterile water, 7.5 μL. PCR amplification conditions were as follows: 1) 95℃, 3 min, pre-denaturation; 2) 95℃, 15 s, denaturation; 3) 60℃, 15 s, annealing; 4) 72℃, 40 s, extension; steps 2-4 were repeated for 30 cycles, with a final extension at 72℃ for 5 min. This confirmed the presence of pMSBBAC1-NT in E. coli ET12567 / pUB307.

[0109] Table 3 Primers used

[0110]

[0111] (4) Inoculate the pMSBBAC1-NT-transferred Escherichia coli ET12567 / pUZ8002 into 5 mL of LB liquid medium containing 50 μg / mL apramycin, 50 μg / mL kanamycin, and 25 μg / mL chloramphenicol, and incubate overnight at 37°C and 220 rpm. The remaining culture steps are the same as in (2), and the bacterial resuspended in 0.5 mL of LB medium for later use.

[0112] (5) The mutant strain QZ102-3 was streaked onto YMG solid medium and cultured at 30℃ for 5–7 days. Spores were collected with 20% glycerol solution and a spore suspension was prepared. 500 μL of the spore suspension was centrifuged at 5000 rpm for 2 min, the supernatant was discarded, and the spores were washed twice with 500 μL of LB medium. The suspension was then resuspended in 500 μL of TSB medium and heat-shocked at 50℃ for 10 min to induce spore germination for later use.

[0113] (6) Mix 100 μL of Escherichia coli ET12567 / PUZ8002 bacterial suspension and 100 μL of spore suspension, spread on MS solid medium, and incubate upside down at 30°C for 20 h. Add nalidixic acid (final concentration 45 μg / mL) and apramycin (50 μg / mL), evenly cover the surface of the solid medium, dry in a clean bench, and continue incubation upside down at 30°C. Single colonies of zygotes can be seen after 3-4 days. Transfer the single colonies to YMG solid medium containing apramycin (final concentration 50 μg / mL) and incubate at 30°C for 5-7 days. Select white single colonies and inoculate them into 15 mL of TSB medium containing 50 μg / mL apramycin. Incubate at 30°C and 220 rpm for 36 h. Harvest mycelium to extract genomic DNA for PCR verification to obtain the engineered Streptomyces chrysosporium.

[0114] (7) The obtained Streptomyces engineered strain was subjected to shake-flask fermentation. The concentration of natamycin in the fermentation broth was detected by HPLC, and a high-natamycin-producing Streptomyces engineered strain QZ103 was finally obtained. The shake-flask fermentation results are as follows: Figure 4 As shown, the fermentation yield of QZ103 reached 6.4 g / L, which was 82.3% higher than that of the mutant strain QZ102-3 and 8.42 times that of the original strain QZ101.

[0115] Strain QZ103 was deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC No. 35422.

[0116] Example 4

[0117] This embodiment provides the application of a high-natamycin-producing engineered Streptomyces strain in natamycin production. In this embodiment, the high-natamycin-producing engineered Streptomyces strain QZ103 was used in a 15L fermenter for production with the starting strain QZ101.

[0118] The specific operation process is as follows:

[0119] (1) Seed culture: The engineered strain QZ103 and the starting strain QZ101, streaked on YMG solid medium containing apramycin (50 μg / mL), were inoculated into 250 mL Erlenmeyer flasks containing 30 mL of seed culture medium, respectively. The cultures were incubated at 30 °C and 230 rpm for 18–24 h until the PMV reached 4–6%, yielding the primary seed culture. The primary seed culture was then inoculated at a rate of 3% into 1 L Erlenmeyer flasks containing 250 mL of seed culture medium, and incubated at 30 °C and 230 rpm for another 18–24 h until the PMV reached 4–6%, yielding the secondary seed culture. The seed culture medium contained 2% glucose, 1.5% peptone, and had a pH of 7.0. The culture was sterilized at 115 °C for 30 min.

[0120] (2) Fermentation Culture: The secondary seed culture obtained in step (1) was transferred to a 15L fermenter at an inoculation rate of 10%. The total volume of the fermentation medium in the fermenter was 10L. The fermenter temperature was controlled at 30℃ and 500rpm, the aeration rate was 1-2v / v / m, and the dissolved oxygen in the fermenter was controlled above 30%. During the fermentation process, the glucose concentration was controlled at 10-20g / L by feeding glucose. When the pH was below 6.0, a 5M sodium hydroxide solution was automatically fed to control the pH at 6.0. If necessary, an antifoaming agent was added to control foam. Fermentation lasted for 7 days. The fermentation medium contained 4% glucose, 2.5% peptone, 1% yeast extract, 0.02% magnesium sulfate heptahydrate, 0.03% calcium carbonate, and a pH of 7.0. It was sterilized at 121℃ for 30 minutes, with glucose added after separate sterilization.

[0121] The results are as follows Figure 5 As shown, the natamycin yield of the starting strain QZ101 was 7.9 g / L, while the fermentation yield of the engineered strain QZ103 reached 29.2 g / L, which was 269% higher than that of the starting strain QZ101.

[0122] The above specific embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

[0123] The specific nucleic acid sequence involved in this invention is as follows:

[0124] SEQ ID NO.1:

[0125] Name: NATA1-F primer

[0126] Sequence type: DNA (other DNA)

[0127] Biological origin: synthetic construct

[0128] CTCGAACCGCCCAAGATGC

[0129] SEQ ID NO.2:

[0130] Name: NATA1-R primers

[0131] Sequence type: DNA (other DNA)

[0132] Biological origin: synthetic construct TGGCGTCGAATTCCTCCGGSEQ ID NO.3:

[0133] Name: NATA2-F primer sequence type: DNA (other DNA) organism source: synthetic construct ACTTCACGAAGTCGTCCACSEQ ID NO.4:

[0134] Name: NATA2-R Primer Sequence Type: DNA (other DNA) Organism Origin: synthetic construct CTTGATAAAACGTTGACCA SEQ ID NO.5:

[0135] Name: pMS-F primer sequence type: DNA (other DNA) Organism origin: synthetic construct CGAGCGACATGATGGGACC SEQ ID NO. 6:

[0136] Name: pMS-R primer sequence type: DNA (other DNA) organism source: synthetic construct CGACAGGTGCTGAAAGCGAG.

Claims

1. A strain of Streptomyces producing high yields of natamycin, characterized in that: The Streptomyces is named Streptomyces gilvosporeus QZ101, and the preservation number is CGMCC No. 35421, and the preservation unit is China General Microbiological Culture Collection Center.

2. A Streptomyces engineered strain with high natamycin production, characterized in that: The Streptomyces engineering strain is obtained by using electron accelerator mutagenesis technology to treat the Streptomyces strain as claimed in claim 1, and then introducing a BAC plasmid containing a natamycin synthesis gene cluster through genetic engineering technology.

3. The engineered Streptomyces bacterium of claim 2, wherein: The natamycin synthesis gene cluster is derived from Streptomyces chattanoogensis.

4. The engineered Streptomyces bacterium of claim 3, wherein: The Streptomyces engineering strain is named Streptomyces gilvosporeus QZ103, and the preservation number is CGMCC No. 35422, and the preservation unit is China General Microbiological Culture Collection Center.

5. A method for constructing an engineered strain of Streptomyces producing high yield of natamycin, characterized in that, The method comprises the following steps: S1, using an electron accelerator to perform at least one round of irradiation mutagenesis treatment on a starting strain to obtain a mutant strain; the starting strain is the Streptomyces strain as claimed in claim 1; during the irradiation mutagenesis treatment, the treatment dose is 1-5 kGY; S2, introducing a BAC plasmid containing a natamycin synthesis gene cluster into the mutant strain to obtain a Streptomyces engineering strain.

6. The method for constructing an engineered strain of Streptomyces producing high amounts of natamycin according to claim 5, characterized in that: In each round of irradiation mutagenesis treatment, multiple gradient-increasing dose groups are selected in the range of 1-5 kGY to perform irradiation treatment on the spore suspension, and after irradiation, multiple single colonies are randomly selected from each dose group for screening.

7. Application of the Streptomyces strain as claimed in claim 1 or the Streptomyces engineering strain as claimed in any one of claims 2-4 in fermentation production of natamycin.

8. A method for fermentative production of natamycin using the Streptomyces strain according to claim 1 or the engineered Streptomyces strain according to any one of claims 2 to 4, characterized in that, The method comprises the following steps: D1, inoculating the strain into a seed culture medium, and culturing in a constant-temperature shaker at a temperature of 28-32 DEG C, so that the cell accumulation volume reaches 4-6%, to obtain a seed liquid; S2, inoculating the seed liquid into a fermentation culture medium at a volume inoculation amount of 3-5%, and fermenting at a temperature of 28-32 DEG C for 120-168 h to obtain a fermentation liquid containing natamycin.

9. The method of claim 8, wherein: The seed culture medium comprises the following components and mass / volume ratios: glucose content of 1-3%, peptone content of 1-3%, NaCl content of 0.25-1%, and pH of 6.0-7.

5.

10. The method of claim 8, wherein: The fermentation culture medium comprises the following components and mass / volume ratios: glucose content of 4-8%, peptone content of 0.5-3%, yeast powder content of 0.5-3%, magnesium sulfate heptahydrate content of 0.05-0.3%, calcium carbonate content of 0.05-0.4%, and pH of 6.0-7.5.

Images