Streptomyces noursei for producing multi-fungal element B and method for increasing yield of multi-fungal element B

Abstract

The invention relates to the technical field of microorganisms, in particular to streptomyces noursei for producing multi-fungal element B and a method for increasing the yield of the multi-fungal element B. According to the streptomyces noursei for producing the multi-fungal element B, negative regulation of the multi-fungal element B is relieved or negative regulation is relieved and positive regulation is enhanced through a combined genetic engineering strategy, so that the yield of the multi-fungal element B is remarkably increased, and byproducts are remarkably reduced; in addition, the strain also has relatively high genetic stability. The method for increasing the yield of the polymycotin B of the streptomyces nosella provides an effective transformation target for construction of a polymycotin B production strain, and has a relatively good application prospect in the production of the polymycotin B. The invention further provides a method for improving the yield of the polymycotin B of the streptomyces nosella.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more particularly to a strain of Streptomyces Norilsk that produces polyfungin B and a method for increasing the yield of polyfungin B. Background Technology

[0002] Polyfungin B is a polyene macrolide antibiotic with broad-spectrum antifungal activity, exhibiting significantly higher antibacterial efficacy than nystatin A1 and A3. Currently, polyfungin B is mainly produced through fermentation of *Streptomyces northerly*, but wild-type strains have low yields (<0.2 mg / mL), and issues such as unclear metabolic regulation mechanisms and insufficient optimization of fermentation processes limit its industrial application.

[0003] Existing technologies for increasing polyfungin B production mainly rely on mutagenesis breeding and culture medium optimization, but these methods suffer from drawbacks such as unclear genetic background and limited yield increases (usually <50%). Research indicates that antibiotic biosynthesis in *Streptomyces northerly* is controlled by a complex regulatory network, including globally negative regulatory genes. nsdA It can inhibit secondary metabolic pathways and positively regulate genes. nysRIV and glycosyltransferase gene nysGtf They respectively participate in the transcriptional activation and post-modification processes of biosynthesis. Summary of the Invention

[0004] To address the problem of low polyfungin B production in existing Streptomyces Norilskia strains, this invention provides a polyfungin B-producing Streptomyces Norilskia strain and a method for increasing polyfungin B production.

[0005] Specifically, the present invention provides the following technical solutions.

[0006] In a first aspect, the present invention provides the application of reducing the expression and / or activity of the globally negative regulatory protein NsdA in improving the production performance of polyfungin B in Streptomyces.

[0007] This invention has found that reducing the expression and / or activity of the globally negative regulatory protein NsdA can enhance the production capacity of polyfungin B in Streptomyces.

[0008] Secondly, the present invention provides the application of reducing the expression and / or activity of the globally negative regulatory protein NsdA and increasing the expression and / or activity of the positive regulatory protein NysRⅣ in improving the production performance of polyfungin B in Streptomyces.

[0009] By synergistically regulating the global negative regulatory protein NsdA and the positive regulatory protein NysRⅣ, the two can work together to more effectively enhance the production capacity of polyfungin B in Streptomyces.

[0010] Thirdly, the present invention provides the application of reducing the expression and / or activity of the globally negative regulatory protein NsdA and increasing the expression and / or activity of NysRⅣ and NysGtf proteins in improving the production performance of polyfungin B in Streptomyces.

[0011] This invention discovers that by simultaneously regulating the global negative regulatory protein NsdA, the positive regulatory protein NysRⅣ, and the glycosyltransferase NysGtf, the synergistic effect of these three can more effectively enhance the polyfungin B production capacity of Streptomyces, and the effect is significantly better than regulating the global negative regulatory protein NsdA alone.

[0012] In the above applications, the Streptomyces is preferably Streptomyces Knowlesi.

[0013] In the above applications, the production performance of polyfungin B includes the yield, conversion rate, and / or production intensity of polyfungin B. The conversion rate is the conversion rate of the substrate to polyfungin B. The production intensity is the yield of polyfungin B per unit time.

[0014] In this invention, the reduction in expression and / or activity can be achieved through any one or more of the following methods: (1) Mutate the amino acid sequence of the protein; (2) Mutate the nucleotide sequence of the protein-coding gene; (3) Replace the transcriptional and / or translational regulatory elements of protein-coding genes with elements that are less active.

[0015] Mutations in the amino acid sequence include deletions, insertions, or substitutions of one or more amino acids. Mutations in the nucleotide sequence include deletions, insertions, or substitutions of one or more nucleotides. The transcriptional and translational regulatory elements include promoters, ribosome binding sites, enhancers, etc.

[0016] Preferably, the reduction in NsdA expression and / or activity is achieved by knocking out... nsdA Genetic implementation.

[0017] In this invention, the enhancement of expression and / or activity can be achieved through any one or more of the following methods: (1) Increase the copy number of protein-coding genes; (2) Replace the transcriptional and / or translational regulatory elements of protein-coding genes with more active elements; (3) Mutate the nucleotide sequence of the protein-coding gene; (4) Mutate the amino acid sequence of the protein.

[0018] Increasing the copy number of a protein-coding gene can be achieved by increasing the copy number of the gene on chromosomes and / or endogenous plasmids, or by introducing an exogenous plasmid containing the gene. The transcriptional and translational regulatory elements include promoters, ribosome binding sites, enhancers, etc. Mutations in the nucleotide sequence include deletions, insertions, or substitutions of one or more nucleotides. Mutations in the amino acid sequence include deletions, insertions, or substitutions of one or more amino acids.

[0019] Preferably, the expression and / or activity enhancement of NysRⅣ and NysGtf proteins are achieved by overexpressing the encoding genes of the proteins.

[0020] Preferably, overexpression of the gene encoding the protein involves increasing the copy number of the gene. This increase in copy number is preferably achieved by introducing a plasmid containing the gene. The plasmid is preferably an integrative plasmid.

[0021] The amino acid sequences and their encoding gene sequences of the NsdA, NysRⅣ, and NysGtf proteins of Streptomyces can be obtained by those skilled in the art through publicly available databases.

[0022] For *Streptomyces northerly*, the amino acid sequence of NsdA is shown in SEQ ID NO.1. The amino acid sequence of NysRⅣ is shown in SEQ ID NO.2, and the amino acid sequence of NysGtf is shown in SEQ ID NO.3.

[0023] Fourthly, the present invention provides a Streptomyces Norilskia that produces polyfungin B, wherein the Streptomyces Norilskia is modified to reduce the expression and / or activity of its globally negative regulatory protein NsdA.

[0024] Furthermore, the Streptomyces Norilskia is modified to enhance the expression and / or activity of its positive regulatory protein NysRⅣ; or, the Streptomyces Norilskia is modified to enhance the expression and / or activity of its NysRⅣ and NysGtf proteins.

[0025] The polyfungin B production of the aforementioned Streptomyces Norilskia bacterium is increased due to the modification.

[0026] For Streptomyces Norilskia, regulating NsdA alone can improve the production performance of polyfungin B. On this basis, combined regulation of NysRⅣ, or combined regulation of NysRⅣ and NysGtf, can further enhance the production performance of polyfungin B of the strain.

[0027] Preferably, the reduction in NsdA expression and / or activity is achieved by knocking out... nsdA Genetic implementation.

[0028] Preferably, the enhanced expression and / or activity of the NysRⅣ protein and NysGtf protein are achieved by overexpressing the encoding genes of the proteins.

[0029] Preferably, overexpression of the gene encoding the protein involves increasing the copy number of the gene. This increase in copy number is preferably achieved by introducing a plasmid containing the gene. The plasmid is preferably an integrative plasmid (e.g., pSET152-KT). The overexpression is preferably an increase in the copy number of the gene within the genome.

[0030] In some embodiments of the present invention, a polyfungin B-producing Streptomyces northerneri is provided, wherein... nsdA The gene was knocked out.

[0031] In other embodiments of the invention, a polyfungin B-producing Streptomyces northerneri is provided, wherein... nsdA The gene was knocked out and overexpressed. nysRIV and nysGtf Genes. This strain works by knocking out globally negative regulatory genes. nsdA and overexpression of positive regulatory genes nysRIV and glycosyltransferase gene nysGtf The combined engineering strategy significantly improved the biosynthetic efficiency of polyfungin B.

[0032] For the polyfungin B-producing Streptomyces Norilskia, the following modifications may be further included: reduced expression and / or activity of the NysL protein.

[0033] Preferably, the reduction in the expression and / or activity of the NysL protein is achieved by knocking out... nysL Genetic implementation.

[0034] The amino acid sequence of the NysL protein is shown in SEQ ID NO.4.

[0035] In some embodiments of the present invention, a polyfungin B-producing Streptomyces northerneri is provided, wherein... nsdA and nysL The gene was knocked out.

[0036] In other embodiments of the invention, a polyfungin B-producing Streptomyces northerneri is provided, wherein... nsdA and nysL The gene was knocked out and overexpressed. nysRIV and nysGtf Gene.

[0037] The present invention does not have any particular limitation on the starting strain of *Streptomyces northerneri* that produces polyfungin B. The starting strain can be any *Streptomyces northerneri* capable of synthesizing polyfungin B. The polyfungin B-producing *Streptomyces northerneri* is constructed by the above modifications, and its polyfungin B production is increased compared with that of the starting strain.

[0038] In some embodiments of the present invention, the polyfungin B-producing Streptomyces Norilskia... nsdA and nysL The gene was knocked out and overexpressed. nysRIV and nysGtf Gene; the strain was named Streptomyces-GBC-Syphu-2025-5C and was deposited on July 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and classified as Streptomyces northerneri. Streptomyces noursei The accession number is CGMCC No.35188.

[0039] Fifthly, the present invention provides any of the following applications of the polyfungin B-producing Streptomyces Norilskia described above: (1) Application in the production of polyfungin B; (2) Application in the construction of production strains of polyfungin B.

[0040] The Streptomyces Norilskia that produces polyfungin B provided by this invention can be used directly for fermentation to produce polyfungin B, or it can be used as a starting strain to construct a polyfungin B production strain.

[0041] In a sixth aspect, the present invention provides a method for producing polyfungin B, the method comprising: culturing the polyfungin B-producing Streptomyces norscheriensis and collecting polyfungin B from the culture.

[0042] Preferably, the fermentation medium used for the culture comprises the following components: glucose 50-60 g / L, soybean meal 20-30 g / L, MgSO4·7H2O 0.2-0.8 g / L, and (NH4)2SO4 2-4 g / L.

[0043] Preferably, the culture temperature is 28~30℃, and / or the pH is 7.0-7.5.

[0044] Preferably, the production method of polyfungin B includes seed culture and fermentation culture.

[0045] In a seventh aspect, the present invention provides a method for increasing the production of polyfungin B in Streptomyces Norilsk, the method comprising: modifying Streptomyces Norilsk to reduce the expression and / or activity of the globally negative regulatory protein NsdA; Alternatively, modify Streptomyces Norilskia to reduce the expression and / or activity of the globally negative regulatory protein NsdA and increase the expression and / or activity of the positive regulatory protein NysRⅣ. Alternatively, *Streptomyces northerly* can be modified to reduce the expression and / or activity of the globally negative regulatory protein NsdA and enhance the expression and / or activity of NysRⅣ and NysGtf proteins.

[0046] The beneficial effects of this invention include at least the following: This invention provides a *Streptomyces northerly* strain that produces polyfungin B. This strain either removes negative regulation of polyfungin B or, through a combination of genetic engineering strategies, removes negative regulation and enhances positive regulation, significantly increasing the yield of polyfungin B. Moreover, by directionally modifying key regulatory genes, metabolic network disorder is effectively avoided, and byproducts (such as 10-deoxynystatin) are significantly reduced. In addition, this strain exhibits high genetic stability; after three consecutive generations, the yield of polyfungin B fluctuates by less than 10%. The method for increasing the yield of polyfungin B in *Streptomyces northerly* provided by this invention significantly improves the biosynthetic efficiency of polyfungin B through a combination of genetic engineering strategies, providing an effective modification target for the construction of polyfungin B-producing strains, and providing efficient strains and key technologies for the industrial production of polyfungin B. Attached Figure Description

[0047] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0048] Figure 1 This is a schematic diagram of the construction process of the genetically engineered strain in Example 1 of the present invention.

[0049] Figure 2 In Embodiment 1 of the present invention nsdA Electrophoresis diagram of PCR verification of gene knockout, where M represents DNA marker; lanes 1-4 are respectively PCR verification of the upstream homologous arm of ΔnysL strain, PCR verification of the upstream homologous arm of ΔnysLΔnsdA strain, PCR verification of the downstream homologous arm of ΔnysL strain, and PCR verification of the downstream homologous arm of ΔnysLΔnsdA strain.

[0050] Figure 3This is a bar chart comparing the yield of polyfungin B and the accumulation of by-products in Example 1 of the present invention (starting strain vs. genetically engineered strain). Detailed Implementation

[0051] In a specific embodiment of the present invention, a polyfungin B-producing Streptomyces norscheri is provided, the construction method of which includes the following steps: (1) Knockout nsdA Gene: The knockout vector pKCNAUD (containing the temperature-sensitive plasmid pKC1139) was constructed using the temperature-sensitive plasmid pKC1139. nsdA (Upstream and downstream homologous arms), the starting strain was introduced via conjugation transfer, and obtained via homologous double exchange. nsdA Knockout strains; (2) Overexpression nysRIV :Will nysRIV The gene was cloned into the integrative vector pSET152-KT to construct pSETRⅣ, which was then conjugated and transferred into the aforementioned nsdA knockout strain to obtain... nsdA Knockout and overexpression nysRIV strains; (3) Overexpression nysGtf :Will nysGtf Gene cloned into pSET152-KT, pSET- nysGtf , joint transfer to the above nsdA Knockout and overexpression nysRIV strains, obtained nsdA Knockout and overexpression nysRIV and nysGtf Engineered strains.

[0052] The starting strain is Streptomyces Norilsk ( Streptomyces noursei GBC-A10 (i.e., Streptomyces Norilsk SN-72) ΔnysL This strain was deposited on May 6, 2023, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China) and classified as *Streptomyces norscheriensis*. Streptomyces noursei The strain, with accession number CGMCC No. 27276, has been disclosed in patent application CN 120249153 A. *Streptomyces northerly* ( Streptomyces noursei GBC-A10 has been removed nysL The gene blocks the synthesis of nystatin A3.

[0053] The above nsdA Knockout and overexpression nysRIV and nysGtfThe engineered strain, fermented under optimized conditions for 72 hours, produced a polyfungin B yield of 0.66 mg / mL, which is 247.37% higher than the original strain, and exhibited good genetic stability.

[0054] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0055] Example 1: nsdA Construction of gene knockout strains Streptomyces norkelsteini ( Streptomyces noursei SN-72 ΔnysL For the starting strain, construct nsdA Gene knockout strains, the specific methods are as follows, flowchart as shown. Figure 1 As shown: Knockout vector construction: using Streptomyces northernii SN-72 ΔnysL Using the genome as a template, amplification was performed using primers NA.UF / NA.UR and NA.DF / NA.DR. nsdA The upstream and downstream homologous arms (2210 bp and 2174 bp) were sequentially cloned into the pKC1139 vector to obtain the knockout vector pKCNAUD.

[0056] Conjugation transfer: The knockout vector pKCNAUD was introduced into E. coli ( Escherichia coli ) ET12567 (pUZ8002) was mixed with a suspension of Streptomyces SN-72ΔnysL spores (donor-recipient ratio 1:1), heat-shocked at 50°C for 10 min, pre-germinated at 37°C for 3 h, then plated on ISP4 plates, and covered with a 50 μg / mL amycin-resistant plate after 22 h to screen for double-exchange strains.

[0057] The above-mentioned combination transfer method is an optimized method, which improves the combination transfer efficiency by 4.12 times compared with the initial conditions.

[0058] Verification: PCR amplification showed nsdA Gene (1485 bp) deletion ( Figure 2 The strain was named Streptomyces Norilsk SN-72. ΔnysLΔnsdA HPLC results showed that... nsdAThe yield of polyfungin B in the gene knockout strain was increased by 126.32% compared to the original strain, reaching 0.43 mg / mL. Furthermore, the accumulation of the byproduct 10-deoxynystatin during fermentation was significantly lower than that of the original strain, *Streptomyces northerneri* SN-72. ΔnysL It decreased by 35.29% ( Figure 3 ).

[0059] Example 2: nysRIV and nysGtf Construction of overexpression strains Streptomyces northerneri SN-72 constructed in Example 1 ΔnysLΔnsdA Based on this, overexpression nysRIV and nysGtf Genes, the specific methods are as follows: Construction of overexpression vectors: Cloning nysRIV (718 bp) and nysGtf (1201 bp) to pSET152-KT, constructing vectors pSETRⅣ and pSET-nysGtf; in, nysRIV The PCR amplification primers for the gene were RⅣ.F (5'-GGAAGATCTCCGGGCGTCACTTGACGAATTC-3', SEQ ID NO.5) and RⅣ.R (5'-GGAAGATCTGATGGCGGTCTCGTGACTATCACTC-3', SEQ ID NO.6), and the amplified fragment size was 718 bp. nysGtf The PCR amplification primers for the gene were Gtf.F (5'-AAGGAAAAAAGCGGCCGCGCCCGTATGCGTGTCCTGCTGATC-3', SEQ ID NO.7) and Gtf.R (5'-GGAAGATCTGTGAGGCTCATGTGCCCGCTC-3', SEQ ID NO.8), and the amplified fragment size was 1201 bp.

[0060] Stepwise conjugation transfer: pSETRⅣ and pSET-nysGtf were sequentially introduced into Streptomyces northerneri SN-72. ΔnysL ΔnsdA Positive clones were selected through apramycin resistance screening; PCR validation: 1270 bp was amplified using specific primers. nysRIV ) and 1339 bp ( nysGtf (fragment, confirmation) nysRIV and nysGtf Gene integration yielded the engineered strain Streptomyces Norilsk SN-72 ΔnysLΔnsdA::nysGtf:: nysRIV .

[0061] Example 3: Fermentation Condition Optimization and Yield Testing For Streptomyces northernii SN-72 ΔnysLΔnsdA::nysGtf::nysRIV The fermentation conditions were optimized as follows: Carbon and nitrogen source screening: When glucose (55 g / L) was used as the carbon source, the yield of polyfungin B increased by 22.3% compared to maltose. When soybean meal (25 g / L) was used as the nitrogen source, byproducts such as 10-deoxynivalenzyme were reduced by 18.7% compared to yeast extract. Therefore, glucose (55 g / L) was determined to be the optimal carbon source, and soybean meal (25 g / L) was determined to be the optimal nitrogen source.

[0062] The final fermentation medium composition was determined as follows: glucose 55 g / L, soybean meal 25 g / L, MgSO4·7H2O 0.5 g / L, and (NH4)2SO4 3 g / L.

[0063] The final culture conditions were: 28℃, 200 rpm shaking culture for 72 h, initial pH 7.5.

[0064] Fermentation experiments were conducted using the final determined fermentation medium and culture conditions, and the yield of polyfungin B was determined. Prior to fermentation, a seed culture was performed. The seed culture medium formula was as follows: soluble starch 10.0 g / L, peanut meal 20.0 g / L, (NH4)2SO4 2.0 g / L, K2HPO4·3H2O 0.2 g / L, Peptone 2.0 g / L, MgSO4·7H2O 0.5 g / L, light CaCO3 6.0 g / L, soybean oil 3.0 g / L, pH 7.2. The bacterial suspension obtained from the seed culture was inoculated into freshly prepared fermentation medium at a ratio of 10% (v / v) for fermentation.

[0065] After fermentation, HPLC analysis showed that the retention time of polyfungin B was 24.63 min, and that the engineered strain *Streptomyces northerly* SN-72... ΔnysLΔnsdA::nysGtf::nysRIV The produced polyfungin B had a purity >95% and a yield of 0.66 mg / mL, which was higher than that of the original strain, *Streptomyces northerly SN-72*. ΔnysL The yield of (0.19 mg / mL) increased by 247.37%.

[0066] The HPLC detection method was as follows: a WondaSil C18 Superb column (5 μm, 4.6 × 250 mm) was used, the mobile phase was acetonitrile-methanol-0.05M sodium acetate buffer (pH 4.0) = 26:37:37 (v / v / v), the flow rate was 0.7 mL / min, the column temperature was 30℃, the detection wavelength was 305 nm, and the retention time of polyfungin B was 24.63 min.

[0067] Example 4: Genetic stability test of engineered strains For Streptomyces northernii SN-72 ΔnysLΔnsdA::nysGtf::nysRIV Genetic stability tests were conducted. The results showed that after three consecutive subcultures, the yields of polyfungin B were 0.66, 0.62, and 0.64 mg / mL, respectively, with an RSD of 3.2%, indicating that the strain had good genetic stability. The fermentation medium and culture conditions used were the same as those determined in Example 2.

[0068] In summary, by knocking out nsdA Remove global negative regulation and synergistic overexpression nysRIV and nysGtf This significantly enhanced the polyfungin B synthesis capacity of *Streptomyces northerly*. The engineered strain achieved a polyfungin B yield of 0.66 mg / mL, which is 3.47 times that of the original strain. Furthermore, the process was stable and controllable, demonstrating significant value for industrial applications.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of reduced expression and / or activity of the globally negative regulatory protein NsdA in improving the production performance of polyfungin B in Streptomyces.

2. Application of reduced expression and / or activity of the globally negative regulatory protein NsdA and enhanced expression and / or activity of the positive regulatory protein NysRⅣ in improving the production performance of polyfungin B in Streptomyces.

3. Application of reduced expression and / or activity of the globally negative regulatory protein NsdA and increased expression and / or activity of NysRⅣ and NysGtf proteins in improving the production performance of polyfungin B in Streptomyces.

4. The application according to any one of claims 1 to 3, characterized in that, The Streptomyces mentioned is Streptomyces Norilsk; And / or, reduced NsdA expression and / or activity through knockout nsdA Genetic realization; And / or, enhanced expression and / or activity of NysRⅣ and NysGtf are achieved by overexpressing the gene encoding the protein.

5. A polyfungin B-producing Streptomyces northerneri, characterized in that, The Streptomyces Norilskia was modified to reduce the expression and / or activity of its globally negative regulatory protein NsdA.

6. The *Streptomyces northerneri* producing polyfungin B according to claim 5, characterized in that, The Streptomyces Norilskia was modified to enhance the expression and / or activity of its positive regulatory protein NysRⅣ; Alternatively, the Streptomyces Norilskia may be modified to enhance the expression and / or activity of its NysRⅣ and NysGtf proteins.

7. The *Streptomyces northerneri* producing polyfungin B according to claim 6, characterized in that, The reduction in NsdA expression and / or activity is achieved through knockout. nsdA Genetic realization; And / or, the enhanced expression and / or activity of the protein is achieved by overexpressing the gene encoding the protein; Preferably, the expression and / or activity of the NysL protein in *Streptomyces northerly* is reduced; the reduction in the expression and / or activity of the NysL protein is preferably achieved by knockout. nysL Genetic realization; Preferably, the polyfungin B-producing Streptomyces Norilskia is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35188.

8. Any of the following applications of the polyfungin B-producing Streptomyces Norilskia as described in any one of claims 5 to 7: (1) Application in the production of polyfungin B; (2) Application in the construction of production strains of polyfungin B.

9. A method for producing polyfungin B, characterized in that, The method comprises: culturing Streptomyces Norilskia as described in any one of claims 5 to 7, and collecting polyfungin B from the culture; Preferably, the fermentation medium used for the culture comprises the following components: glucose 50-60 g / L, soybean meal 20-30 g / L, MgSO4·7H2O 0.2-0.8 g / L, (NH4)2SO4 2-4 g / L; And / or, the culture temperature is 28~30℃, and / or, the pH is 7.0-7.

5.

10. A method for increasing the yield of polyfungin B in Streptomyces northerly, characterized in that, The method includes: modifying Streptomyces Norilskia to reduce the expression and / or activity of the globally negative regulatory protein NsdA; Alternatively, modify Streptomyces Norilskia to reduce the expression and / or activity of the globally negative regulatory protein NsdA and increase the expression and / or activity of the positive regulatory protein NysRⅣ. Alternatively, *Streptomyces northerly* can be modified to reduce the expression and / or activity of the globally negative regulatory protein NsdA and enhance the expression and / or activity of NysRⅣ and NysGtf proteins.

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