Streptomyces gilvosporeus strain with high yield of natamycin and application of streptomyces gilvosporeus strain

Abstract

The invention discloses a streptomyces gilvosporeus strain with high natamycin yield and application thereof, and belongs to the technical field of industrial microorganisms. The phosphate-tolerant natamycin high-yield strain GR-2 is successfully bred by combining protoplast fusion with phosphate tolerance screening, and the yield of natamycin produced by fermentation of the strain in a shake flask can reach 1.81 g / L and is 2.26 times that of an original strain; the fermentation yield in a fermentation tank reaches 13.9 g / L, the passage stability is good, the industrial production requirement can be met, and wide application prospects are achieved.

Description

Technical Field

[0001] The invention relates to a Streptomyces chrysoporus strain with high natamycin production and application thereof, belonging to the technical field of industrial microorganisms. Background Art

[0002] Natamycin is a natural polyene macrolide antibiotic with a chemical formula of C 33 H 47 NO 13 , mainly produced by several Streptomyces (such as Streptomyces natalensis, Streptomyces chatanoogensis and Streptomyces gilvosporeus). Natamycin's unique four-conjugated double-bond twenty-six-membered lactone ring structure enables it to effectively bind to ergosterol in fungal cell membranes at low concentrations, exhibiting strong broad-spectrum antifungal activity. In addition, natamycin is stable in powder form, easily soluble in organic solvents and has no activity loss when stored in the dark. Because of its low toxicity to mammalian cells and no obvious side effects, natamycin has been approved for food preservation and antiseptic use in more than 40 countries. In addition, in the medical field, the antibiotic is widely used in the treatment of antifungal infections and is used as a crop protection agent in cancer treatment and agriculture to prevent fungal contamination. According to statistics, the global annual output value of natamycin has reached US$61 million in 2021, and is expected to exceed US$100 million by 2032. However, despite the huge market demand, the current low industrial production capacity and high production cost of natamycin have limited its commercial promotion to a certain extent. Therefore, improving the synthesis capacity of natamycin production strains has become a hot topic in current research.

[0003] The strain selection methods for natamycin mainly include genetic engineering and random mutagenesis. Although metabolic engineering methods can effectively improve the fermentation performance of strains, it is difficult to accurately locate the key regulatory sites that affect the synthesis of natamycin due to the complex genetic background of Streptomyces chrysogenum. Therefore, traditional mutagenesis methods are still widely used to increase production. Common traditional mutagenesis methods include ultraviolet mutagenesis (UV), nitrosoguanidine (NTG), ethyl methanesulfonate (EMS) and diethyl sulfate (DES) mutagenesis. Although these methods can effectively increase the yield of natamycin, the positive mutation rate is often low. Normal temperature and pressure plasma mutagenesis (ARTP) is an emerging mutagenesis technology in recent years. Its principle is to change the structure of microbial cell membranes through plasma jets, thereby causing gene damage. This technology has the advantages of high mutation rate, significant biological effects, and non-toxicity, showing important application prospects. Compared with other mutagenesis techniques, ARTP can produce mutations in multiple genes at the same time, thereby accelerating the increase in strain yield. DES is a chemical mutagen that reacts with phosphate groups in DNA to cause base mismatches during DNA replication, ultimately inducing mutations. Protoplast fusion technology uses artificial means to achieve genome exchange and recombination, thereby screening out fusion products that have the excellent characteristics of both parents. This technology has a wide range of parent selection and high screening efficiency, which can significantly shorten the time required for strain transformation.

[0004] Phosphorus is an essential element for microbial growth and metabolism. However, natamycin-producing bacteria are extremely sensitive to phosphate. Adding low concentrations of phosphate (2-10 mM) during the fermentation process can inhibit bacterial growth and the expression of the natamycin synthesis gene cluster, resulting in a significant decrease in the production of natamycin. Therefore, by screening natamycin-producing bacteria for phosphate tolerance, the inhibition of phosphate on its synthesis can be relieved, and the ability of the strain to transport and utilize phosphate can be improved, so that it can still grow rapidly and synthesize natamycin in a high concentration phosphate environment.

[0005] In summary, the method of combining protoplast fusion with phosphate tolerance screening can improve cell metabolism and obtain a high-yield natamycin strain. This method is an effective technical means at present and has important practical application value. Summary of the invention

[0006] In response to the problem of low synthesis ability of natamycin producing strains, the present invention provides a strain of Streptomyces gilvosporeus GR-2, which is classified and named Streptomyces gilvosporeus, and has been deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee on September 2, 2024, with a deposit number of CGMCC No.31811, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] In one embodiment, the natamycin high-producing strain can grow in MS medium and cultured at 28°C for 8 to 10 days. The colony diameter is 4 to 6 mm and the color is brown-yellow. The single colony has a regular morphology, is smooth and full, has rounded and complete edges, is slightly concave in the center, has no radial cracks, and has white aerial hyphae growing.

[0008] The invention provides a microbial preparation containing the Streptomyces chrysoporus GR-2.

[0009] In one embodiment, the microbial preparation contains spores of Streptomyces fusiosporum GR-2.

[0010] The invention also provides a method for preparing natamycin by using the Streptomyces chrysoporus GR-2.

[0011] In one embodiment, the method comprises fermenting the Streptomyces chrysosporium GR-2 in a fermentation medium.

[0012] In one embodiment, the fermentation medium contains calcium carbonate, peptone, soluble starch, soybean powder, sodium chloride, yeast powder, and glucose.

[0013] In one embodiment, the method is to inoculate mature spores of Streptomyces chrysoporus into a seed culture medium at an inoculation rate of 4-10%, culture at 28-30°C for 20-30h, then transfer the prepared seed liquid to a fermentation medium, and continue to culture at 28-30°C for at least 96h.

[0014] In one embodiment, the method is to inoculate mature Streptomyces spores into a seed culture medium, culture at 28-30°C for 20-30 hours, then inoculate the seed liquid into a fermentation medium, and continue fermenting at 28-30°C for 96-120 hours.

[0015] In one embodiment, the dissolved oxygen concentration is maintained above 30% during the fermentation process.

[0016] In one embodiment, the fermentation process is further fed to control the residual sugar concentration in the fermentation broth to be between 19.5 and 20.5 g / L.

[0017] The present invention also provides application of the Streptomyces chrysoporus GR-2, or the microbial preparation, or the method in preparing natamycin or a product containing natamycin.

[0018] Beneficial effects:

[0019] (1) The protoplast fusion technology proposed in the present invention is combined with the method of phosphate tolerance screening, which is easy to operate, has a high positive mutation rate, and has a significant yield improvement effect, and is suitable for a wider range of application scenarios.

[0020] (2) The present invention screened out a high-yield strain S.gilvosporeus GR-2 with good spore growth through protoplast fusion technology. The yield of this strain in shake flask fermentation reached 1.81 g / L, which is 2.26 times the yield of the initial strain; the yield in a 5L fermenter reached 13.9 g / L, which was 85.3% higher than the yield of the initial strain (7.5 g / L).

[0021] (3) The strains screened by the present invention have good stability in passage, can meet the needs of industrial applications, and have broad application prospects.

[0022] Biomaterial Deposit

[0023] Streptomyces gilvosporeus GR-2, classified as Streptomyces gilvosporeus, was deposited in the General Microbiology Center of China National Microbiological Culture Collection on September 2, 2024, with the deposit number CGMCC No.31811, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The colony morphology of Streptomyces gilvosporeus ATCC 13326 (S.gilvosporeus ATCC 13326) and GR-2 (S.gilvosporeus GR-2) are shown in the figure; a is a single colony photo of ATCC 13326; b is a single colony photo of GR-2.

[0025] Figure 2 This is the standard curve for determining the yield of natamycin by HPLC.

[0026] Figure 3 This is a graph showing the changing trends of strain biomass and natamycin production during the protoplast fusion combined with phosphate tolerance screening.

[0027] Figure 4This is a comparison chart of parameters for 5L fed-batch fermentation of the original strain S.gilvosporeus ATCC 13326 and the natamycin high-producing strain S.gilvosporeus GR-2. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0029] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0030] Unless otherwise specified, the experimental materials used in the following examples are all conventional biochemical reagents.

[0031] (I) Determination of Natamycin Production (HPLC Method):

[0032] Establishment of Natamycin standard curve:

[0033] Weigh 0.020 g of natamycin standard and dissolve it completely in 100 mL of 5% glacial acetic acid methanol in the dark (heat to 40°C in the dark if necessary) to prepare a natamycin standard solution with a mother liquor concentration of 0.2 g / L. Dilute the mother liquor into standard solutions with concentrations of 0.15, 0.10, 0.075, 0.05, 0.035, and 0.025 g / L, respectively. Sterilize by filtering through a 0.22 μm organic membrane and set aside. Use the HPLC method to draw a standard curve with the peak area as the ordinate and the mass concentration of natamycin (C) as the abscissa (see Figure 2 ).

[0034] Method for determining the yield of natamycin: Take 9 mL of 5% glacial acetic acid methanol and 1 mL of fermentation broth, add them to a 15 mL light-proof centrifuge tube. Use a vortex instrument to oscillate and mix for 2 minutes, and then centrifuge at 4°C and 4200 rpm for 20 minutes. Take 4 mL of the supernatant and put it into a 5 mL light-proof centrifuge tube. Use a 1 mL syringe to filter through an organic membrane and set aside for later use. Use HPLC to determine the concentration of natamycin, and calculate the content of natamycin according to the standard curve.

[0035]

[0036] (II) Biomass determination method:

[0037] Take 4 mL of the fermentation broth in a 5 mL centrifuge tube, centrifuge at 12000 rpm for 10 min, filter with a dried and weighed filter paper, and collect the Streptomyces chrysogenum cells. Dry the cells in a 105°C oven overnight to a constant weight, then weigh and calculate the dry weight of the cells.

[0038] (III) Culture medium:

[0039] MS medium: mannitol 20.0 g / L, soybean powder 20.0 g / L, agar 20.0 g / L.

[0040] Seed culture medium: yeast powder 10.0g / L, glucose 60.0g / L, ammonium sulfate 15.0g / L, potassium dihydrogen phosphate 3.0g / L, dipotassium hydrogen phosphate 1.6g / L, magnesium sulfate 1g / L, zinc sulfate 0.04g / L, iron sulfate 0.03g / L, pH 6.8-7.0.

[0041] Fermentation medium: calcium carbonate 10.0 g / L, peptone 8.0 g / L, soluble starch 50.0 g / L, soybean powder 20.0 g / L, sodium chloride 3.0 g / L, yeast powder 8.0 g / L, glucose 35.0 g / L, pH 6.8-7.0.

[0042] Solid regeneration medium: peptone 4.5g / L, sucrose 118g / L, calcium chloride 0.4g / L, glucose 25g / L, magnesium chloride 10g / L, agar 20g / L, trace element solution 2mL / L, TES buffer (pH 7.5) 10mL / L, pH 6.8-7.0. Trace element solution: ZnCl2 40mg / L, Na2B4O7·10H2O 10mg / L, FeCl3·6H2O 200mg / L, MnCl2·4H2O10mg / L, CuCl2·2H2O 10mg / L, (NH4)6Mo7O 24 4H2O 10mg / L.

[0043] Example 1 Screening of phosphate-tolerant high-yield mutants using ARTP

[0044] a) Take S.gilvosporeus ATCC 13326 as the starting strain, culture it in the seed culture medium at 28-30℃ for 20-30h, collect the bacteria, prepare the spore suspension, and use ARTP mutagenesis for 90s. Take 100μL of the diluted mutagenic bacterial suspension, spread it on MS culture medium containing 1.6g / L KH2PO4, and culture it at 28℃ for 4d. Randomly select 100 single colonies for expansion culture in MS culture medium containing 1.6g / L KH2PO4, and use agar column method for initial screening. The indicator bacteria is Saccharomyces cerevisiae, and the indicator bacteria culture medium (YPD): peptone 20g / L, glucose 20g / L, yeast powder 10g / L, pH 6.0. Biological detection medium: The upper layer is YPD culture medium with 15g / L agar powder added, containing a certain concentration of sensitive bacteria, and the final concentration in the culture medium reaches 1×10 7 CFU·mL -1The lower layer was YPD medium supplemented with 20 g / L agar powder. Six mutants with the largest diameter of inhibition zone were selected for shake flask fermentation, and the concentration of natamycin in the fermentation broth was detected by HPLC, and finally the mutant AP-1 with the highest yield in the first round of ARTP mutagenesis was obtained.

[0045] b) Using AP-1 obtained in step a) as the starting strain, a spore suspension was prepared, and a second round of ARTP mutagenesis was carried out according to the method in step a), and the suspension was spread on MS medium containing 3.2 g / L phosphate. After culturing at 28°C for 4 days, 100 single colonies were randomly selected for expansion and preliminary screening was performed using the agar column method. The six mutant strains with the largest inhibition zone diameter were selected for shake flask fermentation, and the concentration of natamycin in the fermentation broth was detected by HPLC, and finally the mutant strain AP-2 with the highest yield in the second round of ARTP mutagenesis was obtained.

[0046] c) Using AP-2 obtained in step b) as the starting strain, a spore suspension was prepared, and the third round of ARTP mutagenesis was carried out according to the method described in step a), and the suspension was coated on MS medium containing 5.6 g / L phosphate. The suspension was cultured at 28°C for 4 days, 100 single colonies were randomly selected for expansion, and preliminary screening was performed using the agar column method. The six mutant strains with the largest inhibition zone diameter were selected for shake flask fermentation, and the concentration of natamycin in the fermentation broth was detected by HPLC, and finally the mutant strain AP-3 with the highest yield in the third round of ARTP mutagenesis was obtained.

[0047] Example 2 Screening of phosphate-tolerant mutants using DES

[0048] A) Spore suspension was prepared using S.gilvosporeus ATCC 13326 as the starting strain, and treated with diethyl sulfate (DES) at a final concentration of 1% for 40 minutes. 100 μL of the diluted mutagenic bacterial suspension was spread on MS medium containing 1.6 g / L KH2PO4 and cultured at 28°C for 4 days. 100 single colonies were randomly selected and cultured according to the method of step (1), and preliminary screening was performed using the agar column method. The six mutant strains with the largest inhibition zone diameter were selected for shake flask fermentation, and the concentration of natamycin in the fermentation broth was detected by HPLC, and finally the mutant strain DP-1 with the highest yield of the first round of DES mutagenesis was obtained.

[0049] B) Using the strain DP-1 obtained in step (A) as the starting strain to prepare a spore suspension, the method of step a) was used to carry out the second round of DES mutagenesis. Take 100 μL of the diluted mutagenic bacterial suspension, apply it to MS medium containing 3.2 g / L KH2PO4, and culture it at 28°C for 4 days. Randomly select 100 single colonies for expansion culture and perform preliminary screening using the agar column method. Select the six mutant strains with the largest diameter of the inhibition zone for shake flask fermentation, and use HPLC to detect the concentration of natamycin in the fermentation broth, and finally obtain the mutant strain DP-2 with the highest yield of the second round of DES mutagenesis.

[0050] C) Prepare a spore suspension using the strain DP-2 obtained in step (B) as the starting strain, and perform the third round of DES mutagenesis using the method of step a). Take 100 μL of the diluted mutagenic bacterial suspension, apply it to MS medium containing 5.6 g / L KH2PO4, and culture it at 28°C for 4 days. Randomly select 100 single colonies for expansion and perform preliminary screening using the agar column method. Select the six mutants with the largest inhibition zone diameter for shake flask fermentation, and use HPLC to detect the concentration of natamycin in the fermentation broth, and finally obtain the mutant DP-3 with the highest yield in the third round of DES mutagenesis.

[0051] Example 3 Screening of Phosphate-tolerant Natamycin High-producing Bacteria

[0052] The mutant strain AP-3 screened in the above Example 1 and the mutant strain DP-3 screened in Example 2 were subjected to protoplast preparation, fusion and protoplast regeneration: First, mycelium culture and protoplast preparation were performed on AP-3 and DP-3 respectively. Subsequently, the protoplast solution of AP-3 was subjected to 15W ultraviolet irradiation for 120s for ultraviolet inactivation; the protoplast solution of DP-3 was heated in a 70°C water bath for 30min for heat inactivation. Then, the two inactivated parent strains were subjected to protoplast fusion and spread on a resistance plate containing KH2PO4 for culture. Select full colonies with good spore growth, and perform primary screening by agar column method and secondary screening by shake flask in turn. Finally, a high-yield natamycin mutant strain GR-2 was obtained on a resistance gradient plate containing 0-4g / L KH2PO4. Compared with the original strain, GR-2 mycelium grew faster and the colony morphology was smoother and fuller (see Figure 1 ).

[0053] The natamycin production capacity of the highly phosphate-tolerant Streptomyces chrysoporus GR-2 was determined. Two rings of spores were scraped from the MS medium plate full of spores and inoculated into a 250mL Erlenmeyer flask containing 30mL of seed medium and cultured at 28°C for 24±4h. Subsequently, the seed liquid was inoculated into a 250mL Erlenmeyer flask containing 30mL of fermentation medium at a 6% inoculation rate and cultured at 28°C for 96±6h. After the culture was completed, samples were taken and the natamycin yield and biomass were determined according to determination method I and determination method II, respectively.

[0054] like Figure 3 As shown in the figure, after ARTP and DES mutagenesis, the yield of natamycin gradually increased with the increase of the number of mutagenesis. Through protoplast fusion, the yield of AP-3 and DP-3 was further improved, and the yield of the obtained mutant in shake flask fermentation reached 1.81±0.03g / L, which was 2.26 times that of the original strain. The mutant was named S.gilvosporeus GR-2, and the strain was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number CGMCCNo.31811.

[0055] The phosphate tolerance of the starting strain Streptomyces chrysoporus ATCC 13326 and the phosphate-tolerant Streptomyces chrysoporus GR-2 was compared. The two strains were cultured on resistance gradient plates containing 0-4g / L KH2PO4 at 28℃ for 4 days, and the growth of the two strains was observed. Compared with the starting strain Streptomyces chrysoporus ATCC 13326, GR-2 showed a significant growth advantage in high phosphate medium, and its cell density and growth rate were higher than those of ATCC 13326.

[0056] The two strains were inoculated into shake flask fermentation medium containing 5.6g / L KH2PO4, cultured at 28℃ and 220rpm for 4 days, and the natamycin production in the fermentation broth was determined. Compared with the starting strain Streptomyces chrysospora ATCC 13326, the natamycin production of GR-2 was 1.60±0.10g / L, which was 2 times higher.

[0057] Example 4 Stability verification of strain GR-2

[0058] This example mainly investigates and verifies the stability of the high-yield mutant strains. The selected high-yield mutant strains are continuously plate-passaged. The high-yield strain GR-2 is inoculated on MS solid culture medium and cultured in a 28°C incubator for 7 days. The strain is picked and inoculated into a new MS solid culture medium and continuously passaged for more than 8 times.

[0059] The strains of each generation (F1-F8) were inoculated into the shake flask fermentation medium with an inoculation spatula, and cultured at 28°C and 220 rpm for 96-120 h. The yield of natamycin in the fermentation broth was detected, and the results are shown in Table 1. The high-yield natamycin mutant strain GR-2 obtained by three rounds of protoplast fusion iterative composite mutagenesis combined with phosphate tolerance screening had good genetic stability within 8 generations, and the yield was maintained at 1.80±0.10 g / L.

[0060] Table 1 Results of the subculture stability test of the mutant strain GR-2

[0061] Subculture strains Natamycin yield (g / L) F1 1.81 F2 1.88 F3 1.85 F4 1.72 F5 1.75 F6 1.70 F7 1.77 F8 1.80

[0062] Example 5 Preparation of Natamycin by a Fed-Batch Method Using Streptomyces chrysoporus GR-2 with High Natamycin Production

[0063] Preparation of seed solution: The original strain (S.gilvosporeus) ATCC 13326 and the high-yield strain (S.gilvosporeus) GR-2 streaked on the MS plate were inoculated into 30 mL of sterilized seed culture medium. The culture medium was placed on a shaker and cultured at 28°C and 200 rpm for 30 hours to obtain the seed solution of ATCC 13326 and GR-2.

[0064] Fermentation culture: ATCC 13326 and GR-2 seed liquid were inoculated into a 5L fermenter containing 3.5L fermentation medium at an inoculum rate of 6% (V / V) for fed-batch fermentation. The dissolved oxygen was maintained at more than 30% by controlling the changes in ventilation and rotation speed. When the pH dropped below 6.0, a 25% ammonia solution (V / V) was automatically added to control the pH at 6.0. When the glucose concentration in the fermentation broth was lower than 20g / L, 500g / L of glucose mother solution was automatically added to control the sugar concentration at about 20±0.5g / L, and the fermentation was continued for 120h to finally obtain the fermentation broth.

[0065] Determine the natamycin production and bacterial count in the fermentation broth. Figure 4 As shown in the figure: The bacterial mass of both strains grew rapidly at the beginning of fermentation, but showed different growth trends in the later stage. The bacterial mass of ATCC 13326 slowed down after 48 hours of fermentation, and the final bacterial mass dry weight was 31.5g / L; GR-2 grew slowly after 72 hours of fermentation, and the final bacterial mass dry weight reached 39.2g / L. The natamycin production of the two strains continued to accumulate throughout the fermentation process. After 120 hours of fermentation, the natamycin production of the original strain ATCC 13326 was 7.5g / L, while the production of the high-yield mutant GR-2 reached 13.9g / L, which was 85.3% higher than that of the original strain.

[0066] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Streptomyces gilvosporeus GR-2 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 2, 2024, with the deposit number CGMCC No.31811.

2. A microbial preparation containing the Streptomyces chrysoporus GR-2 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The microbial preparation contains spores of Streptomyces chrysoporus GR-2.

4. A method for preparing natamycin, characterized in that: The Streptomyces chrysosporus GR-2 according to claim 1 or the microbial preparation according to any one of claims 2 to 3 is fermented in a fermentation medium.

5. The method according to claim 4, characterized in that The fermentation medium contains calcium carbonate, peptone, soluble starch, soybean powder, sodium chloride, yeast powder and glucose.

6. The method according to claim 5, characterized in that The spores of Streptomyces chrysoporus GR-2 are cultured in a seed culture medium at 28-30° C. for 20-30 hours to prepare a seed solution, and then the seed solution is transferred to a fermentation culture medium for fermentation.

7. The method according to any one of claims 4 to 6, characterized in that: The fermentation is carried out at 28-30° C. for at least 96 hours.

8. The method according to claim 7, characterized in that During the fermentation process, the dissolved oxygen concentration was maintained above 30%.

9. The method according to claims 4 to 8, characterized in that: The fermentation process is also fed, and the feeding includes but is not limited to the addition of glucose.

10. Use of the Streptomyces chrysoporus GR-2 according to claim 1, or the microbial preparation according to any one of claims 2 to 3, or the method according to any one of claims 4 to 9 in the preparation of natamycin or a product containing natamycin.

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