Application of phosphite in promoting streptomyces to generate antibiotics

Abstract

The invention relates to application of phosphite in promoting streptomyces to produce antibiotics. Specifically, the invention relates to application of phosphite in promoting streptomyces to generate antibiotics and a method for promoting streptomyces to generate antibiotics. According to the present invention, the phosphite is added to the fermentation basal culture medium to induce the streptomyces to produce the antibiotics, and the streptomyces is promoted to produce the antibiotics, especially the polyketone antibiotics. The method is simple to operate and convenient to use, and has a relatively good application prospect.

Description

Technical Field

[0001] The present disclosure relates to the field of microbial technology, and in particular, to the application of phosphite in promoting Streptomyces to produce antibiotics. Background Art

[0002] Streptomyces is one of the main bacterial species that produce antibiotics. The antibiotics it produces have wide application value in many fields such as medicine and agriculture. At present, about two-thirds of antibiotics are produced by microorganisms of this genus, so Streptomyces is called a natural cell factory for drug synthesis. However, the level of antibiotic synthesis of wild Streptomyces isolated from nature is very low, which is difficult to meet the requirements of industrialization; the already industrialized engineered strains also need to continuously increase production to reduce production costs. At present, antibiotic synthesis is mainly enhanced through gene mutagenesis and gene editing. However, gene mutagenesis has problems such as blindness, large workload, and genetic instability. Gene editing requires the construction of a strain genetic operation system, which not only takes a lot of time, but also not every type of Streptomyces can construct a genetic operation system. In addition, gene editing also requires the accumulation of necessary expression control elements, which further increases the difficulty of using gene editing to enhance antibiotic synthesis.

[0003] Phosphite is usually used as a plant stimulant to enhance plant immunity. Streptomyces has a good tolerance to phosphite, but some Gram-negative bacteria are more sensitive to it. Applying phosphite to the soil can enhance the competitiveness of Streptomyces in the microbial community and resist the growth of pathogens. However, it is not clear whether phosphite can increase the production of more antibiotics by Streptomyces.

[0004] Therefore, research that can increase the production of antibiotics synthesized by Streptomyces is of great significance. Summary of the invention

[0005] The purpose of the present disclosure is to provide an application of phosphite in improving the antibiotic production capacity of Streptomyces and a method for improving the antibiotic production capacity of Streptomyces. Phosphite, especially potassium phosphite, can further promote the production of antibiotics by Streptomyces, and the method is simple to operate and easy to use.

[0006] In order to achieve the above objectives, the present disclosure provides, in a first aspect, the use of phosphite in promoting the production of antibiotics by Streptomyces.

[0007] Optionally, the Streptomyces is selected from at least one of the deposit numbers CGMCC No.32501, CGMCC No.32502 and CGMCC No.32503.

[0008] Optionally, the phosphite is selected from at least one of potassium phosphite, sodium phosphite, calcium phosphite and ammonium phosphite.

[0009] Optionally, the antibiotic comprises a polyketide antibiotic.

[0010] A second aspect of the present disclosure provides a method for promoting Streptomyces to produce antibiotics, the method comprising: inoculating Streptomyces into a fermentation basal medium and adding phosphite, and culturing the medium in a shaker.

[0011] Optionally, the content of the phosphite is 0.001-0.003 g per 1 mL of the fermentation basal medium; After adding phosphite, the pH value of the fermentation basal medium is 6-7.

[0012] Optionally, the phosphite is selected from at least one of potassium phosphite, sodium phosphite, calcium phosphite and ammonium phosphite.

[0013] Optionally, the fermentation basal medium is selected from at least one of Gao's medium No. 1, PDA medium, LB medium, ISP medium, NA medium, hygroscopic Streptomyces medium, oat medium, MS medium, a medium with glycerol as the carbon source, and a medium with molasses as the carbon source.

[0014] Optionally, the Streptomyces is selected from at least one of the deposit numbers CGMCC No.32501, CGMCC No.32502 and CGMCC No.32503.

[0015] Optionally, the culture conditions include: a temperature of 26-30° C., a rotation speed of 150-200 rpm, and a time of 5-10 days.

[0016] Through the above technical solution, the present invention adds phosphite to the fermentation basal medium to induce Streptomyces to produce antibiotics, thereby promoting Streptomyces to produce antibiotics, especially polyketide antibiotics. The method is simple to operate, easy to use, and has good application prospects.

[0017] Other features and advantages of the present disclosure will be described in detail in the following detailed description.

[0018] Biomaterial Deposit The Streptomyces loucheii (SSW1B2410) disclosed in the present invention is a pure culture isolated from a Streptomyces microbial fertilizer product by the inventor of the present invention, and its preservation number is CGMCC No.32501, and the preservation date is November 6, 2024. The preservation unit is the General Microbiological Center of the China Microbiological Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and is classified and named Streptomyces rochei .

[0019] The heat-spined spore Streptomyces (SSW2FA16) disclosed in the present invention is a pure culture isolated by the inventor of the present invention from field soil in Nanjing, and its preservation number is CGMCC No.32502, and the preservation date is November 6, 2024. The preservation unit is the General Microbiological Center of the China Microbiological Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and is classified and named Streptomyces thermospinosisporus .

[0020] The heat-spined spore Streptomyces (SSW3FA19) disclosed in the present invention is a pure culture isolated by the inventor of the present invention from field soil in Nanjing, and its preservation number is CGMCC No.32503, and the preservation date is November 6, 2024. The preservation unit is the General Microbiological Center of the China Microbiological Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and is classified and named Streptomyces thermospinosisporus . BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings are used to provide a further understanding of the present disclosure and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present disclosure but do not constitute a limitation of the present disclosure. In the accompanying drawings: Figure 1 This is a plate diagram of antibiotic production by Streptomyces in the presence of potassium phosphite in Example 1 of the present disclosure.

[0022] Figure 2 This is a plate diagram of antibiotic production by Streptomyces in the presence of potassium phosphite in Example 2 of the present disclosure.

[0023] Figure 3 It is the expression level of polyketide synthesis genes in Streptomyces under the condition of potassium phosphite in Example 1. DETAILED DESCRIPTION

[0024] The specific implementation of the present disclosure is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described herein is only used to illustrate and explain the present disclosure, and is not used to limit the present disclosure.

[0025] A first aspect of the present disclosure provides use of phosphite in promoting the production of antibiotics by Streptomyces.

[0026] In the present disclosure, the inventors of the present disclosure unexpectedly discovered that when Streptomyces is cultured in the presence of phosphite, phosphite may induce Streptomyces to up-regulate genes related to antibiotic synthesis, thereby prompting Streptomyces to synthesize antibiotics.

[0027] According to the present disclosure, the Streptomyces is selected from at least one of the deposit numbers CGMCC No.32501, CGMCC No.32502 and CGMCC No.32503.

[0028] Among them, the Streptomyces with the preservation number of CGMCC No.32501 (Streptomyces loucheri, SSW1B2410) was isolated from the Streptomyces microbial fertilizer product, and its taxonomic name is Streptomyces rochei ; Streptomyces with the deposit number CGMCC No.32502 (Streptomyces thermosporus, SSW2FA16) was isolated from field soil in Nanjing and its taxonomic name is Streptomyces thermospinosisporus ; Streptomyces with the deposit number CGMCC No.32503 (Streptomyces thermosporus, SSW3FA19) was isolated from field soil in Nanjing and its taxonomic name is Streptomyces thermospinosisporus .

[0029] According to the present disclosure, the phosphite is selected from at least one of potassium phosphite, sodium phosphite, calcium phosphite and ammonium phosphite; preferably potassium phosphite.

[0030] According to the present disclosure, the antibiotics include polyketide antibiotics. Preferably, the polyketide antibiotics include synthetic PKS type I antibiotics and synthetic PKS type II antibiotics.

[0031] A second aspect of the present disclosure provides a method for promoting Streptomyces to produce antibiotics, the method comprising: inoculating Streptomyces into a fermentation basal medium and adding phosphite, and culturing the medium in a shaker.

[0032] In one embodiment of the present disclosure, the content of the phosphite is 0.001-0.003 g per 1 mL of the fermentation basal medium; after adding the phosphite, the pH value of the fermentation basal medium is 6-7. When the content of the phosphite is within the range of the above embodiment, it can further promote the production of antibiotics by Streptomyces; if the content of the phosphite is too low, its induction ability to Streptomyces is limited; if the content of the phosphite is high, the growth of the Streptomyces cells is severely inhibited.

[0033] According to the present disclosure, the phosphite is selected from at least one of potassium phosphite, sodium phosphite, calcium phosphite and ammonium phosphite.

[0034] According to the present disclosure, the fermentation basal medium is selected from at least one of Gao's medium No. 1, PDA medium, LB medium, ISP medium, NA medium, hygroscopic Streptomyces medium, oat medium, MS medium, a medium with glycerol as a carbon source, and a medium with molasses as a carbon source.

[0035] According to the present disclosure, the Streptomyces is selected from at least one of the deposit numbers CGMCC No.32501, CGMCC No.32502 and CGMCC No.32503.

[0036] In one embodiment of the present disclosure, the culture conditions include: temperature of 26-30°C, rotation speed of 150-200 rpm, and time of 5-10 d; preferably, the culture conditions include: temperature of 28°C, rotation speed of 170 rpm, and time of 10 d.

[0037] The present disclosure is further illustrated below by way of examples, but the present disclosure is not limited thereby.

[0038] Experimental Materials: Phosphorous acid: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0039] Potassium hydroxide: purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0040] Gao's medium: Haibo Biotechnology.

[0041] PDA culture medium: Beijing Solebow Technology Co., Ltd.

[0042] Oxford Cup: Higher Education Research (Beijing) Technology Co., Ltd.

[0043] Experimental strains: 1. Streptomyces rochei , the preservation number is CGMCC No.32501 (isolated from Streptomyces microbial fertilizer product).

[0044] 2. Streptomyces thermospinosisporus , the deposit number is CGMCC No.32502 (isolated from field soil in Nanjing area).

[0045] 3. Streptomyces thermospinosisporus , the deposit number is CGMCC No.32503 (isolated from field soil in Nanjing area).

[0046] 4. The pathogen tested is the pathogen of tomato wilt Fusarium oxysporums , (isolated from diseased tomato plants in Hebei, gifted by the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences).

[0047] Example 1 (1) Prepare potassium phosphite mother solution by using 2 g of phosphorous acid, adding 5 mL of distilled water, and then adding 2.2 g of potassium phosphite. Dissolve and adjust the volume to 10 mL. Then filter the mother solution through a 0.25 μm sterile filter membrane to sterilize.

[0048] (2) Cultivation of strains: Inoculate 3 types of Streptomyces stored in a -80°C refrigerator into 5 mL of Goethe's medium respectively, and place in a shaker at 28°C, 170 rpm. After 5 days, take 1 mL of each of the three types of Streptomyces and inoculate into 15 mL of Goethe's medium. Add 0.23 mL of sterile potassium phosphite stock solution to the medium so that the potassium phosphite content in each mL of Goethe's medium is 0.003 g.

[0049] An equal amount of sterile distilled water was added to the control group.

[0050] The cells were then placed in a shaker at 28°C, 170 rpm, and cultured for 10 days.

[0051] Example 2 The same method as in Example 1 was followed, except that 0.08 mL of sterile potassium phosphite stock solution was added to the culture medium so that the content of potassium phosphite in each mL of Gobber's medium was 0.001 g.

[0052] Test Example 1 In this experiment, the size of the inhibition zone of the fermentation broth was observed by the tube-plate method, which indicated the content of antibiotics. 5 CFU / mL Fusarium oxysporum The spore suspension is spread on the PAD medium, and a sterilized Oxford cup is placed on the PAD medium; The first group of tests was as follows: 50 μL of the control group fermentation broth (CK group, containing only Streptomyces fermentation broth), the potassium phosphite treatment group (KP group, no Streptomyces were cultured in the culture medium, and each 1 mL of Gould's culture medium contained 0.0067 g of potassium phosphite), and the potassium phosphite and strain group in Example 1 (CK+KP group, each 1 mL of Gould's culture medium contained 0.0067 g of potassium phosphite and Streptomyces were co-cultured in the culture medium) were placed in an Oxford cup, and after culturing in a 28°C incubator for 7 days, the antibacterial radius was measured, and each treatment was repeated 3 times. The results are as follows: Figure 1 shown.

[0053] The second test group was as follows: 50 μL of the control group fermentation broth (CK group, containing only Streptomyces fermentation broth), potassium phosphite treatment group (KP group, no Streptomyces were cultured in the culture medium, 0.0023 g of potassium phosphite was contained in each 1 mL of Gould's culture medium), and potassium phosphite and strain group in Example 1 (CK+KP group, 0.0023 g of potassium phosphite and Streptomyces were co-cultured in the culture medium in each 1 mL of Gould's culture medium) were placed in an Oxford cup, cultured in a 28°C incubator for 7 days, and then the antibacterial radius was measured. Each treatment was repeated 3 times. The results are as follows: Figure 2 shown.

[0054] Depend on Figure 1It can be seen that the CK group and the KP group could hardly inhibit the growth of pathogens, while the CK+KP group significantly enhanced the ability to inhibit pathogens, indicating that phosphite can promote the production of antibiotics in the Streptomyces fermentation broth.

[0055] Depend on Figure 2 It can be seen that the CK group and the KP group could hardly inhibit the growth of pathogens, while the CK+KP group had a slight ability to inhibit pathogens.

[0056] Example 3 Extraction of the control and potassium phosphite treated samples of Example 1 Streptomyces rochei RNA was extracted from the sample and converted into cDNA. The synthetase fragments of the main antibiotics (polyketide antibiotics) produced by Streptomyces (PKS1: synthesizing PKS type I antibiotics, PKS2: synthesizing PKS type II antibiotics) were amplified by fluorescent quantitative PCR to test whether potassium phosphite enhances the ability of Streptomyces to synthesize the relevant antibiotics. The results are as follows Figure 3 shown.

[0057] Depend on Figure 3 It can be seen that phosphite significantly upregulated the expression of genes related to the synthesis of polyketide substances in Streptomyces, which indicates that phosphite mainly enhances the ability of Streptomyces to produce polyketide antibiotics.

[0058] The preferred embodiments of the present disclosure are described in detail above in conjunction with the accompanying drawings; however, the present disclosure is not limited to the specific details in the above embodiments. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0059] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0060] In addition, various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. The application of phosphite in promoting the production of antibiotics by Streptomyces.

2. The use according to claim 1, wherein: The streptomyces is selected from at least one of the deposit numbers CGMCC No.32501, CGMCC No.32502 and CGMCC No.32503.

3. The use according to claim 1, wherein: The phosphite is selected from at least one of potassium phosphite, sodium phosphite, calcium phosphite and ammonium phosphite.

4. The use according to claim 1, wherein: The antibiotics include polyketide antibiotics.

5. A method for promoting Streptomyces to produce antibiotics, characterized in that: The method comprises: inoculating streptomyces into a fermentation basic medium, adding phosphite, and placing the medium in a shaking table for culturing.

6. The method according to claim 5, wherein: The content of the phosphite is 0.001-0.003 g per 1 mL of the fermentation basal medium; After adding phosphite, the pH value of the fermentation basal medium is 6-7.

7. The method according to claim 6, wherein: The phosphite is selected from at least one of potassium phosphite, sodium phosphite, calcium phosphite and ammonium phosphite.

8. The method according to claim 5, wherein: The fermentation basal medium is selected from at least one of Gao's medium No. 1, PDA medium, LB medium, ISP medium, NA medium, Streptomyces hygroscopicus medium, oat medium, MS medium, a medium with glycerol as a carbon source, and a medium with molasses as a carbon source.

9. The method according to claim 5, wherein: The streptomyces is selected from at least one of the deposit numbers CGMCC No.32501, CGMCC No.32502 and CGMCC No.32503.

10. The method according to claim 5, wherein: The culture conditions include: a temperature of 26-30° C., a rotation speed of 150-200 rpm, and a time of 5-10 days.

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