Streptomyces noursei capable of producing multi-fungal element B and application of streptomyces noursei

Abstract

The invention relates to the technical field of bioengineering, in particular to streptomyces noursei capable of producing multimycotin B and application of the streptomyces noursei. According to the invention, gene editing is carried out on the nysL gene and the nysGtf gene or a combination thereof in the streptomyces nosella, so that the yield of the polymycotin B in the streptomyces nosella is remarkably improved. According to the streptomyces noursei for producing the multi-fungal element B, the multi-fungal element B can be efficiently synthesized and accumulated by utilizing low-cost raw materials, the yield of the multi-fungal element B is remarkably increased, and the streptomyces noursei can be used for producing the multi-fungal element B or increasing the content ratio of the multi-fungal element B in prepared nystatin. According to the streptomyces noursei and the method for producing the multi-fungal element B by using the streptomyces noursei, provided by the invention, a strain resource and an effective method are provided for producing the multi-fungal element B and producing nysfungin with a high multi-fungal element B content ratio, and the streptomyces noursei has a relatively good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly to Streptomyces nogalater producing polyfungin B and its applications. Background Art

[0002] Polyfungin B is one of the active components of the polyene macrolide antifungal antibiotic nystatin, and is a secondary metabolite produced by Streptomyces. Nystatin belongs to multi-component antibiotics and has a broad-spectrum antifungal effect. It is mainly used for the treatment of digestive tract fungal infections and external skin fungal infections, and there are also research reports in the prevention and control of agricultural diseases. The main components of nystatin are nystatin A1, nystatin A3 and polyfungin B. The three have the same structural framework, and the main difference lies in the hydroxyl group at the C-10 position and the digitoxose at the C-35 position. Nystatin A1 and nystatin A3 have one more hydroxyl group at the C-10 position than polyfungin B, and nystatin A3 and polyfungin B have one more digitoxose at the C-35 position than nystatin A1. At present, there is no literature reporting the biosynthetic pathway of nystatin A3 and polyfungin B. Therefore, it is of great significance to explore the synthesis of polyfungin B and nystatin A3 and the functions of related genes. At present, there is no report on constructing a strain with high yield of polyfungin B by genetic engineering means. Summary of the Invention

[0003] The present invention provides Streptomyces nogalater producing polyfungin B and its applications.

[0004] To construct Streptomyces nogalater with high yield of polyfungin B ( Streptomyces noursei ), the present invention has studied the genes that can promote the synthesis and accumulation of polyfungin B, and found that nysL the reduction of the expression and activity of the nysGtf gene can significantly promote the increase in the yield of polyfungin B. On this basis, further increasing the

[0005] expression and / or activity of the gene can further significantly promote the increase in the yield of polyfungin B.

[0005] Specifically, the present invention provides the following technical solutions: In the first aspect, the present invention provides the application of the reduction of the expression of the nysL gene and / or the activity of its encoded protein in improving the production performance of polyfungin B by Streptomyces nogalater.

[0006] In the second aspect, the present invention provides the application of the reduction of the expression of the nysL gene and / or the activity of its encoded protein and nysGtf the increase of the expression of the

[0007] In some embodiments of the present invention, by reducing nysL the expression of the gene and / or the activity of its encoded protein, the production performance of polyfungin B in Streptomyces nogalater is improved.

[0008] In other embodiments of the present invention, by reducing nysL the expression of the gene and / or the activity of its encoded protein while increasing nysGtf the expression of the gene and / or the activity of its encoded protein, the production performance of polyfungin B in Streptomyces nogalater is improved.

[0009] The present invention discovers that nysL when the genes and nysGtf genes are jointly modified as described above, the modifications of the two genes can act synergistically, and the effect is significantly better than that of each single-gene modification.

[0010] In the present invention, the production performance of polyfungin B includes the yield, conversion rate, and / or production intensity of polyfungin B.

[0011] Among them, the conversion rate is the conversion rate from the substrate to polyfungin B. The production intensity is the yield of polyfungin B per unit time.

[0012] In the present invention, there are no special limitations on the technical means for achieving reduced expression and / or activity. For example, common genetic engineering means and gene editing methods can be used to modify the gene, its encoded protein, its regulatory elements, or the protein, so as to reduce the expression of the gene and / or the activity of its encoded protein.

[0013] Exemplarily, the reduction of the expression of the gene and / or the activity of its encoded protein is achieved by any one or more combinations of the following (1) to (3): (1) Mutating the amino acid sequence of the protein; (2) Mutating the nucleotide sequence of the gene; (3) Replacing the transcriptional and / or translational regulatory elements of the gene with elements having weaker activity.

[0014] The above-mentioned mutation of the amino acid sequence includes deletion, insertion, or substitution of one or more amino acids.

[0015] The above-mentioned mutation of the nucleotide sequence includes deletion, insertion, or substitution of one or more nucleotides.

[0016] The above-mentioned transcriptional and translational regulatory elements include promoters, ribosome binding sites, etc.

[0017] In some embodiments of the present invention, nysL the reduction of the expression of the gene and / or the activity of its encoded protein is achieved by knocking out nysLGene implementation.

[0018] In the present invention, there are no special restrictions on the technical means for realizing the expression of a gene and / or enhancing the activity of its encoded protein. For example, common genetic engineering means and gene editing methods can be used to modify the gene, its encoded protein, its regulatory elements or proteins, so as to enhance the expression of the gene and / or the activity of its encoded protein.

[0019] Exemplarily, the increase in the expression of the gene and / or the activity of its encoded protein is achieved by any one or a combination of the following (1) to (4): (1) Increasing the copy number of the gene; (2) Replacing the transcriptional regulatory element and / or translational regulatory element of the gene with a more active element; (3) Mutating the nucleotide sequence of the gene; (4) Mutating the amino acid sequence of the protein.

[0020] The above-mentioned increase in the copy number of the gene can be achieved by increasing the copy number of the gene on the chromosome and / or the endogenous plasmid, or by introducing an exogenous plasmid containing the gene.

[0021] The above-mentioned transcriptional and translational regulatory elements include promoters, ribosome binding sites, etc.

[0022] The above-mentioned mutation of the nucleotide sequence includes deleting, inserting or replacing one or more nucleotides.

[0023] The above-mentioned mutation of the amino acid sequence includes deleting, inserting or replacing one or more amino acids.

[0024] In some embodiments of the present invention, nysGtf the increase in the expression of the gene and / or the activity of its encoded protein is achieved by increasing nysGtf the copy number of the gene, preferably by introducing a plasmid carrying nysGtf the gene.

[0025] In the present invention, the nysL amino acid sequence of the encoded protein of the gene is the amino acid sequence encoded by the sequence shown in SEQ ID NO.1. The nysGtf amino acid sequence of the encoded protein of the gene is the amino acid sequence encoded by the sequence shown in SEQ ID NO.2.

[0026] Thirdly, the present invention provides Streptomyces nogalater that produces polyfungin B, and the Streptomyces nogalater is modified so that nysL the expression of the gene and / or the activity of its encoded protein is reduced; or, the Streptomyces nogalater is modified so thatnysL The expression of the gene and / or the activity of its encoded protein is decreased, while nysGtf the expression of the gene and / or the activity of its encoded protein is increased.

[0027] For nysL the technical means and examples for decreasing the expression of the gene and / or the activity of its encoded protein, nysGtf the technical means and examples for increasing the expression of the gene and / or the activity of its encoded protein, and their preferred methods are as described in the first and second aspects above.

[0028] In some embodiments of the present invention, the Streptomyces noursei is modified to knockout nysL the gene.

[0029] In some embodiments of the present invention, the Streptomyces noursei is modified to knockout nysL the gene, while overexpressing nysGtf the gene.

[0030] The above overexpression is achieved by introducing a plasmid carrying nysGtf the gene.

[0031] Preferably, the amino acid sequence of the encoded protein of the nysL gene is the amino acid sequence encoded by the sequence shown in SEQ ID NO.1; the amino acid sequence of the encoded protein of the nysGtf gene is the amino acid sequence encoded by the sequence shown in SEQ ID NO.2.

[0032] In some embodiments of the present invention, the strain obtained by modifying the Streptomyces noursei to knockout nysL the gene is Streptomyces noursei ( Streptomyces noursei ) GBC-A10. This strain was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (abbreviation: CGMCC, address: No. 3, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code 100101) on May 6, 2023, and was taxonomically named Streptomyces noursei, with the deposit number CGMCC No. 27276.

[0033] The polyfungin B yield of the above-mentioned Streptomyces noursei producing polyfungin B is increased due to the above modification.

[0034] For the starting strain of Streptomyces noursei producing polyfungin B, the present invention has no special limitation. Theoretically, starting from Streptomyces noursei capable of synthesizing polyfungin B, the above-mentioned Streptomyces noursei producing polyfungin B can be constructed through the above modification, and its polyfungin B yield should be increased compared with the starting strain.

[0035] Fourthly, the present invention provides a microbial preparation, which comprises Streptomyces nogalater producing polyfungin B as described above.

[0036] Preferably, in the microbial preparation, Streptomyces nogalater producing polyfungin B exists in the form of viable bacteria.

[0037] The microbial preparation can be a liquid preparation or a solid preparation.

[0038] In addition to Streptomyces nogalater producing polyfungin B, the microbial preparation may further comprise excipients allowed in the field of microbial preparations, including but not limited to carriers, lyoprotectants, etc.

[0039] The above-mentioned microbial preparation can be prepared by conventional technical means, with or without adding excipients allowed in the field of microbial preparations.

[0040] The present invention also provides a preparation method of the above-mentioned microbial preparation, the method comprising: culturing Streptomyces nogalater producing polyfungin B. The culturing is preferably carried out at 25-30 °C.

[0041] Fifthly, the present invention provides any one of the following applications of Streptomyces nogalater producing polyfungin B or the microbial preparation as described above: (1) Application in the production of polyfungin B or nystatin; (2) Application in the preparation of products having a fungal inhibitory function; (3) Application in the breeding of production strains of polyfungin B or nystatin.

[0042] In the above (1), polyfungin B is one of the active ingredients of nystatin, and Streptomyces nogalater can synthesize other active ingredients of nystatin (nystatin A1, nystatin A3). Therefore, Streptomyces nogalater producing polyfungin B can be used for the production of polyfungin B or nystatin.

[0043] In the above (2), the fungi include but are not limited to yeasts (such as Saccharomyces cerevisiae, etc.), Candida, Cryptococcus neoformans, Aspergillus, Mucor, Microsporum, Histoplasma capsulatum, Blastomyces dermatitidis, and dermatophytes, etc.

[0044] Sixthly, the present invention provides a production method of polyfungin B or nystatin, the method comprising: culturing Streptomyces nogalater producing polyfungin B as described above, and collecting polyfungin B or nystatin from the culture.

[0045] Preferably, the culture medium for the culture comprises the following components: 44.0 - 55.0 g / L of glucose, 15.0 - 25.0 g / L of peanut cake powder, 1.0 - 10.0 g / L of silkworm chrysalis powder, 2.0 - 4.0 g / L of peptone, 1.0 - 2.0 g / L of soybean oil, 5.0 - 15.0 g / L of calcium carbonate, 2.0 - 4.0 g / L of ammonium sulfate, 0.01 - 0.05 g / L of potassium dihydrogen phosphate, and 0.3 - 0.6 g / L of magnesium sulfate heptahydrate.

[0046] Preferably, the culture temperature is 28 - 30 °C, and / or the pH is 6.5 - 7.0.

[0047] In some embodiments of the present invention, the method for producing polyfungin B or nystatin comprises the following steps: (1) Strain activation: Inoculating the Streptomyces noursei producing polyfungin B on an activation medium and culturing at 28 - 30 °C for 5 - 7 days; (2) Seed culture: Inoculating the activated Streptomyces noursei in step (1) into a liquid seed medium and culturing at 28 - 30 °C for 18 - 30 h; (3) Fermentation culture: Inoculating the seed liquid obtained in step (2) into a fermentation medium and culturing under the conditions of 28 - 30 °C and pH 6.5 - 7.0.

[0048] In a seventh aspect, the present invention provides a method for increasing the yield of polyfungin B of Streptomyces noursei, the method comprising: modifying Streptomyces noursei such that nysL the expression of the gene and / or the activity of its encoded protein is reduced; or, modifying Streptomyces noursei such that nysL the expression of the gene and / or the activity of its encoded protein is reduced and nysGtf the expression of the gene and / or the activity of its encoded protein is increased.

[0049] For nysL the reduction of the expression of the gene and / or the activity of its encoded protein, nysGtf the technical means for increasing the expression of the gene and / or the activity of its encoded protein, their examples, and their preferred methods are as described in the first aspect above.

[0050] The beneficial effects of the present invention at least include: By knocking out the nysL gene in Streptomyces noursei or nysL the nysGtfThe genes were combinatorially modified, significantly improving the yield of fungichromin B in Streptomyces nogalater; the constructed Streptomyces nogalater producing fungichromin B can efficiently synthesize and accumulate fungichromin B using low-cost raw materials, significantly increasing the yield of fungichromin B, and can be used for the production of fungichromin B or to increase the proportion of fungichromin B in the prepared nystatin. The Streptomyces nogalater provided by the present invention and the method for producing fungichromin B using this strain provide strain resources and effective methods for the production of fungichromin B and the production of nystatin with a high proportion of fungichromin B, and have good application prospects. Description of the Drawings

[0051] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0052] Figure 1 For Example 1 of the present invention nysL PCR verification diagram of the gene disruption strain. Among them, M1 and M2 are DNA markers, lane 1 is the PCR product of the upstream verification primer of the starting strain; lane 2 is the PCR product of the downstream verification primer of the starting strain; lane 3 is the PCR product of the upstream verification primer of the CGMCC No. 27276 strain; lane 4 is the PCR product of the downstream verification primer of the CGMCC No. 27276 strain.

[0053] Figure 2 Schematic diagram of the genotype of the nysL gene double-exchange disruption strain in Example 1 of the present invention.

[0054] Figure 3 For Example 3 of the present invention nysL Gene disruption strain S. noursei Δ nysL HPLC analysis results of the refined fermentation product; among them, a is the nystatin A1 standard; b is the nystatin standard; c is the fermentation product of the starting strain; d is S. noursei Fermentation product of ΔnysL.

[0055] Figure 4 For Example 3 of the present invention nysL Gene disruption strain S. nourseiΔnysL Mass spectrometry analysis results of the refined fermentation product, among which, a and b are S.noursei Mass spectrometry diagram of product 1 (10-deoxynystatin) of the ΔnysL strain, c is S.noursei Mass spectrometry diagram of product 2 (fungichromin B) of the ΔnysL strain.

[0056] Figure 5 In Example 3 of the present invention S. noursei Δ nysL Analysis results of HPLC detection peak areas of the refined fermentation product

[0057] Figure 6 In Example 3 of the present invention S. noursei Δ nysL Analysis results of the proportion of each component content of the refined fermentation product Detailed implementation manners

[0058] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings in the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts shall fall within the protection scope of the present invention

[0059] In the detailed implementation manners of the present invention, Streptomyces noursei 72-22-1 is used as the starting strain, and by blocking (knocking out), complementing and overexpressing its nysL genes, the C-10 hydroxylation function of the nysL gene is verified, and the influence of the nysL gene on the synthesis of nystatin A3 and multifungin B is explored

[0060] Specifically, the present invention successfully constructs a nysL blocking plasmid and nysL blocked Streptomyces noursei S. noursei Δ nysL . Compared with the starting strain, the fermentation product of Streptomyces noursei S. noursei Δ nysL changes from four components to two components, and the yields and component percentage proportions of multifungin B and 10-deoxynystatin are significantly increased, indicating that the yield of multifungin B is significantly increased by modifying the nysL gene

[0061] To further prove the effectiveness of the above modification, a nysL complementing plasmid and nysL complemented strain S.nourseiΔ nysL::nysL are constructed. The results show that the ability of the complemented strain S.nourseiΔnysL::nysL to produce nystatin A1 and nystatin A3 is restored, and the yields of 10-deoxynystatin and multifungin B are reduced, indicating that the product change in Streptomyces noursei S. nourseiΔnysL is indeed caused by the nysL blockade, confirming the nysLDirect influence of genes on the production of nystatin A3 and multifungin B.

[0062] Based on the above results, the present invention verified nysL the C-10 hydroxylation function of the gene, and proved that 10-deoxynystatin can be converted into nystatin A1 through C10 hydroxylation, and multifungin B can be converted into nystatin A3 through C10 hydroxylation.

[0063] On the other hand, in the specific embodiments of the present invention, taking Streptomyces noursei 72-22-1 as the starting strain, by blocking (knocking out), complementing and overexpressing its nysGtf gene, the function of the nysGtf gene was verified, and the influence of the nysGtf gene on the synthesis of nystatin A3 and multifungin B was explored.

[0064] To verify the role of the nysGtf gene in the synthesis of multifungin B and nystatin A3, a nysGtf blocking plasmid and nysGtf blocking strain S. nourseiΔnysGtf were constructed. Compared with the starting strain, S. nourseiΔnysGtf the fermentation products changed from four components to three components, the percentage content and peak area of nystatin A1 and 10-deoxynystatin increased significantly, while the percentage content and peak area of multifungin B decreased significantly.

[0065] Furthermore, a nysGtf complementing plasmid and nysGtf complementing strain S.nourseiΔnysGtf::nysGtf were constructed. Through the analysis of the fermentation products, S.nourseiΔnysGtf::nysGtf the ability to produce nystatin A3 was restored, the yields of nystatin A1 and 10-deoxynystatin decreased, and the yield of multifungin B increased.

[0066] Based on the above results, the present invention verified nysGtf that the gene is involved in the synthesis of digitoxose sugar at C-35 position in multifungin B and nystatin A3, and proved that nystatin A1 can be converted into nystatin A3 through C-35 glycosylation, and 10-deoxynystatin can be converted into multifungin B through C-35 glycosylation.

[0067] In the specific embodiments of the present invention, the strain nysL with the S. noursei Δ nysL gene blocked only produced 10-deoxynystatin and multifungin B components during the fermentation process (the peak area of multifungin B increased from 18576.30 to 36983.80, an increase of 99.09%, and the statistical difference was determined by T-test, P<0.01, which was extremely significant). Overexpressing nysGtfStrains of genes S. noursei::nysGtf In the fermentation test, the peak area of nystatin A3 increased from 16552.20 to 59197.90, an increase of 257.63%, and the peak area of multifungin B decreased from 18576.30 to 17300.20, a decrease of 6.87%. It is proved that overexpressing nysGtf genes is more conducive to the conversion of nystatin A1 to nystatin A3 and is not conducive to the production of multifungin B.

[0068] In the specific implementation manner of the present invention, using S.nourseiΔnysL as the starting strain, by overexpressing nysGtf genes, strain S.nourseiΔnysL::nysGtf is constructed. On the premise of blocking nysL genes to reduce the yield of nystatin A1, overexpressing nysGtf genes makes the common precursor substances of nystatin A1 and multifungin B tend to synthesize the metabolic flux of multifungin B, so as to achieve the effect of further increasing the yield of multifungin B.

[0069] To make the purpose, technical solution and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0070] The formula of the liquid seed medium used in the following examples is as follows: starch 5 - 15 g / L, calcium carbonate 5 - 7 g / L, glucose 5 - 15 g / L, ammonium sulfate 1 - 3 g / L, peanut cake powder 15 - 25 g / L, potassium dihydrogen phosphate 0.1 - 0.3 g / L, peptone 1 - 3 g / L, soybean oil 2 - 4 g / L, magnesium sulfate heptahydrate 0.4 - 0.6 g / L, pH 6.8 - 7.4.

[0071] The formula of the fermentation medium used in the following examples is as follows: glucose 44.0 g / L, peanut cake powder 15.0 g / L, silkworm chrysalis powder 2.0 g / L, peptone 3.0 g / L, soybean oil 1.0 g / L, calcium carbonate 10.0 g / L, ammonium sulfate 3.0 g / L, potassium dihydrogen phosphate 0.02 g / L, magnesium sulfate heptahydrate 0.5 g / L, pH 6.5.

[0072] Example 1: nysL Construction of gene-blocked strain S. noursei CGMCC No. 27276 In this example, using S. noursei 72 - 22 - 1 as the starting strain, blocking thenysL Gene, construction nysL Gene-blocking strain S. noursei ΔnysL , the specific method is as follows: 1. Construction of Escherichia coli ( Escherichia coli ) ET12567 (pUZ8002, pKC1139-LUD) donor strain Competent cells were prepared by the CaCl2 method of Escherichia coli and DNA transformation was carried out. The nysL gene-blocking plasmid pKC1139-LUD (containing the upstream and downstream homologous arms of the nysL gene) was transformed into E. coli ET12567 (pUZ8002), and the triple-antibiotic-resistant strain of chloramphenicol, kanamycin and apramycin was screened to obtain the donor strain for nysL gene blocking E. coli ET12567 (pUZ8002, pKC1139-LUD).

[0073] 2. Construction of the blocking strain S. noursei ΔnysL Using the general method of conjugation transfer, the pKC1139-LUD plasmid in E. coli ET12567 (pUZ8002, pKC1139-LUD) was transferred into S. noursei 72-22-1 to construct nysL the gene-blocking strain S. noursei ΔnysL , Figure 1 is nysL the PCR verification result of the blocking strain, Figure 2 is the genotype diagram of this blocking strain.

[0074] The above nysL gene-blocking strain S. noursei ΔnysL was named GBC-A10 and was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (abbreviation: CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code 100101) on May 6, 2023. The taxonomic name was Streptomyces noursei Streptomyces noursei , and the deposit number was CGMCC No. 27276.

[0075] Example 2: Strain fermentation and product extraction Select a fresh slant of the strain constructed in Example 1, and using aseptic operation techniques, inoculate one loop into 50 mL of liquid seed medium for fermentation, at 28 °C, 150 r·min S. noursei ΔnysL , culture for 24 h. Inoculate the seed liquid into 50 mL / 250 mL of fermentation medium at a transfer inoculation amount of 10%, at 28 °C, 200 r·min -1 , culture for 24 h. Inoculate the seed liquid into 50 mL / 250 mL of fermentation medium at a transfer inoculation amount of 10%, at 28 °C, 200 r·min​-1 , incubate for 86 h, and reserve the cultured fermentation broth for use.

[0076] Since nystatin exists in the mycelium, it needs to be extracted with 98% ethanol to obtain the crude nystatin extract. Since the crude extract contains fermentation by-products such as cycloheximide, further purification is required to obtain the pure product.

[0077] The steps of the product extraction and purification method are as follows: 1. Preparation of crude extract of fermentation product (1) Obtain wet mycelium by centrifugation: Centrifuge the fermentation broth at 3500 r·min -1 for 5 min to obtain wet mycelium.

[0078] (2) Extraction: Extract with 3 times the wet mycelium (W / V) of 98% ethanol, stir for 40 min, and centrifuge at 3500 r·min -1 for 10 min, and then extract the mycelium residue twice with 98% ethanol to obtain the extract.

[0079] (3) Concentration: Combine the extracts and concentrate them to 15% of the original volume under reduced pressure (700 mmHg) to obtain the concentrated solution.

[0080] 2. Purification of fermentation product Store the concentrated solution prepared in 1 above at 4 °C for 24 h to complete crystallization, centrifuge to remove the mother liquor, wash the obtained crystals with ethyl acetate and physiological saline, and finally wash once with ethyl acetate and dry to obtain the purified fermentation product, and store it in the dark for use.

[0081] Example 3: High performance liquid chromatography (HPLC) detection, high performance liquid chromatography - mass spectrometry (HPLC-MS) detection and HPLC-MS semi-preparative liquid chromatography of fermentation products 1. High performance liquid chromatography (HPLC) detection Take 50.0 mg of the purified fermentation product prepared by the method of Example 2 and dissolve it in 5.0 mL of methanol, and perform detection using HPLC. The HPLC detection conditions are as follows: Chromatographic column: Shimadzu C18 Superb (250×4.6 mm, 5 μm); Mobile phase: methanol: acetonitrile: 0.05 M sodium acetate buffer solution (pH 4.0) = 37:26:37 (V:V:V); Flow rate: 1 ml / min; Injection volume: 10 µL; Detection wavelength: 305 nm; Column temperature: 30 °C.

[0082] 2. High performance liquid chromatography - mass spectrometry (HPLC-MS) detection 25.0 mg of the refined fermentation product prepared by the method of Example 2 was dissolved in 5.0 mL of methanol and detected by HPLC. HPLC conditions: chromatographic column: Unitary C18 (250×4.6 mm, 5 μm); mobile phase: methanol: water = 70:30 (V:V); flow rate: 1 ml / min; injection volume: 10 μL; detection wavelength: 305 nm; column temperature: 30 °C.

[0083] MS conditions: electrospray ionization source (ESI), full scan mode (Full Scan); cone voltage 15 V, capillary voltage 0.8 kV.

[0084] 3. HPLC-MS semi-preparative liquid chromatography of fermentation products The refined fermentation product prepared by the method of Example 2 was loaded onto a semi-preparative liquid chromatography column: SinoChrom ODS-BP (5 μm × 10 mm × 250 mm); mobile phase: methanol: water = 70:30 (V:V); flow rate: 4 mL / min; injection volume: 200 μL; ultraviolet detection wavelength: 305 nm; column temperature: 30 °C.

[0085] Using the above method, HPLC analysis was performed on the refined fermentation products of the starting strain and nysL gene-blocked strain S. noursei ΔnysL The results are as follows Figure 3 shown. The HPLC-MS detection results are as follows Figure 4 shown. By examining the changes in the proportion and value of the HPLC peak area of each component of the refined fermentation product, the effect of blocking nysL gene on the production of nystatin by Streptomyces noursei was investigated, and the results are as follows Figure 5 and Figure 6 .

[0086] The results showed that, compared with the starting strain, nysL gene-blocked strain S. noursei ΔnysL only produced 10-deoxynystatin and pimaricin B components, and no longer produced nystatin A1 component and nystatin A3 component. The proportion of 10-deoxynystatin increased from 11.49% to 45.24%. Statistical differences were determined by T-test, P P < 0.01, which was highly significant; the proportion of pimaricin B increased from 32.17% to 45.24%. Statistical differences were determined by T-test, P < 0.01, which was highly significant.

[0087] The above results indicate that nysL gene blocking can significantly increase the production of pimaricin B by Streptomyces noursei.

[0088] Example 4 nysGtf Construction of gene overexpression strain Construct nysGtf a gene overexpression plasmid, which is based on the pSET152 plasmid and introduced with nysGtf a gene, and the expression of the gene is driven by the Kasop* promoter. Transfer the nysGtf gene overexpression plasmid into nysGtf the S.noursei 72-22-1 strain by conjugation to construct S. noursei :: nysGtf strain. The fermentation results show that the percentage content of natamycin B decreases from 32.17% to 21.88%, but the percentage content of nystatin A3 increases from 28.66% to 73.00%. It can be seen that overexpressing the nysGtf gene alone does not promote the increase of natamycin B.

[0089] In S. noursei Δ nysL (with the preservation number of CGMCC No. 27276) strain, overexpress the nysGtf gene. Specifically, transfer the above nysGtf gene overexpression plasmid into S. noursei Δ nysL strain by conjugation to construct S. noursei Δ nysL :: nysGtf . The fermentation results show that the percentage content of natamycin B increases from 45.24% to 50.85%, proving that nysL knockout and nysGtf overexpression combination has a better effect on improving natamycin B.

[0090] The above fermentation method is carried out with reference to the method in Example 2, and the detection method of the product is carried out with reference to the method in Example 3.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. nysL Use of reduction in gene expression and / or activity of its encoded protein in improving the production performance of polyfungin B by Streptomyces nogalater.

2. nysL Reduction in the expression of a gene and / or the activity of its encoded protein, and nysGtf Use of an increase in the expression of a gene and / or the activity of its encoded protein in improving the production performance of polyfungin B by Streptomyces nogalater.

3. Streptomyces nogalater producing natamycin B, characterized in that, The Streptomyces nogalater is modified such that nysL the expression of the gene and / or the activity of its encoded protein is reduced; Alternatively, the Streptomyces noursei is modified so that nysL The expression of the gene and / or the activity of its encoded protein is reduced. nysGtf The expression of a gene and / or the activity of its encoded protein is increased.

4. The Streptomyces nogalater producing natamycin B according to claim 3, characterized in that, The Streptomyces nogalater is modified to knockout nysL genes; Alternatively, the S. knowlesi is modified to knock out nysL Genes, overexpressed nysGtf Gene; Preferably, the nysL amino acid sequence of the encoded protein of the gene is the amino acid sequence encoded by the sequence shown in SEQ ID NO.1; the nysGtf amino acid sequence of the encoded protein of the gene is the amino acid sequence encoded by the sequence shown in SEQ ID NO.2; Preferably, the Streptomyces nogalater is modified by knocking out nysL gene, and the obtained strain is Streptomyces nogalater ( Streptomyces noursei ) GBC-A10, which is deposited in the China General Microbiological Culture Collection Center with the deposit number of CGMCC No. 27276.

5. The Streptomyces nogalater producing polyoxin B according to claim 3 or 4, characterized in that, The nystatin B production of Streptomyces noursei producing nystatin B is increased due to the modification.

6. A microbial preparation, characterized in that, The microbial preparation contains Streptomyces noursei producing nystatin B according to any one of claims 3 to 5.

7. The following any one of the applications of Streptomyces noursei producing nystatin B according to any one of claims 3 to 5 or the microbial preparation according to claim 6: (1) Application in the production of nystatin B or nystatin; (2) Application in the preparation of products with antifungal functions; (3) Application in the breeding of production strains of nystatin B or nystatin.

8. A method for producing natamycin B or nystatin, characterized in that, The method includes: culturing Streptomyces noursei producing nystatin B according to any one of claims 3 to 5, and collecting nystatin B or nystatin from the culture.

9. The method according to claim 8, wherein The culture medium used for the culture includes the following components: glucose 44.0 - 55.0 g / L, peanut cake powder 15.0 - 25.0 g / L, silkworm chrysalis powder 1.0 - 10.0 g / L, peptone 2.0 - 4.0 g / L, soybean oil 1.0 - 2.0 g / L, calcium carbonate 5.0 - 15.0 g / L, ammonium sulfate 2.0 - 4.0 g / L, potassium dihydrogen phosphate 0.01 - 0.05 g / L, magnesium sulfate heptahydrate 0.3 - 0.6 g / L; And / or, the temperature of the culture is 28 - 30 °C, and / or, the pH is 6.5 - 7.

0.

10. A method for increasing the yield of polyfungin B in Streptomyces nogalater, characterized in that, The method includes: modifying Streptomyces nogalater such that nysL the expression of the gene and / or the activity of its encoded protein is reduced; Alternatively, the method comprises: modifying Streptomyces nogalater such that nysL the expression of the gene and / or the activity of its encoded protein is reduced and nysGtf the expression of the gene and / or the activity of its encoded protein is increased.

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