Recombinant genetically engineered bacterium for producing micafungin precursor FR901379 and application of recombinant genetically engineered bacterium

Abstract

The invention discloses a recombinant genetically engineered bacterium for producing a micafungin precursor FR901379 and an application of the recombinant genetically engineered bacterium. The recombinant genetically engineered bacterium for producing the micafungin precursor FR901379 is obtained by performing overexpression on an epigenetic modification factor in a phomopsis sheathing genome and performing screening to obtain the recombinant genetically engineered bacterium for producing the micafungin precursor FR901379. The epigenetic modification factor comprises a histone methyltransferase (Dot 1), a histone methyltransferase (Set2) or a histone deacetylase (Rpd3). The yield of FR901379 produced by the engineering strain is increased by 40% compared with that of an original strain. The method disclosed by the invention has the characteristics of simplicity and convenience in operation, high transformation efficiency and good genetic stability.

Description

(I) Technical Field

[0001] This invention belongs to the field of microbial and genetic engineering technology, and relates to a recombinant genetically engineered bacterium that produces micafungin precursor FR901379, its construction method, and its application. (II) Background Technology

[0002] Echinocandins work by non-competitively inhibiting β-1,3-glucan synthase, a key enzyme in fungal cell wall synthesis, thereby blocking the formation of 1,3-β-D-glucan and leading to fungal cell death. Because human cells lack a cell wall structure, this unique mechanism gives echinocandins a significant advantage in terms of low toxicity and high safety in humans. Currently, the main echinocandins widely used in clinical practice include caspofungin, micafungin, and anidulafungin.

[0003] The production of micafungin depends on its key precursor compound FR901379. It has been reported that the filamentous fungus *Pseudomonas sheathiformis* (…) Coleophoma empetri The precursor can be synthesized. However, the metabolic network and regulatory mechanisms related to the biosynthesis of FR901379 are highly complex, which seriously hinders the targeted modification of the metabolic engineering of *Pittosporum tobira*.

[0004] Epigenetic modification can heritably regulate gene expression without altering the DNA sequence. It significantly affects the physiological phenotype and secondary metabolite biosynthesis of filamentous fungi by changing chromatin state and transcription factor binding capacity. However, current non-directional modification techniques using epigenetic genes to regulate the fermentation synthesis of FR901379 face several technical challenges: firstly, there is a lack of publicly reported effective and predictable regulatory strategies; secondly, due to the inherent complexity of epigenetic regulatory networks, the results of such non-directional modifications are often highly uncertain, making it difficult to achieve targeted and efficient increases in FR901379 yield. Therefore, non-directional modification of epigenetic genes for regulating FR901379 biosynthesis is highly complex and unpredictable. (III) Summary of the Invention

[0005] The purpose of this invention is to provide a recombinant genetically engineered bacterium that produces the micaflavin precursor FR901379 and its applications. This invention achieves this by overexpressing epigenetic modifying factors (histone methyltransferases) in the host bacterium *Pteris vittata*. Dot1 Histone methyltransferase Set2 or histone deacetylase Rpd3 Metabolic network perturbation was implemented to increase the amount of FR901379 produced by overexpressing engineered bacteria through fermentation. At the same time, engineered bacterial strains with significantly improved FR901379 synthesis ability were screened to establish a cell factory for efficient synthesis of FR901379.

[0006] The technical solution adopted in this invention is:

[0007] This invention provides a recombinant genetically engineered bacterium that produces the precursor of micaflavin FR901379, wherein the recombinant genetically engineered bacterium is produced by *Pteris vittata* (a type of fungus). Coleophoma empetri Recombinant genetically engineered bacteria that overexpressed epigenetic modifiers in their genome and screened for high-yield micafungin precursor FR901379 were obtained; the epigenetic modifiers included histone methyltransferases. Dot1 Histone methyltransferase Set2 or histone deacetylase Rpd3 .

[0008] Furthermore, the histone methyltransferase Dot1 The nucleotide sequence is shown in SEQ ID NO.2; the histone methyltransferase Set2 The nucleotide sequence is shown in SEQ ID NO.3; the histone deacetylase Rpd3 The nucleotide sequence is shown in SEQ ID NO.4.

[0009] Furthermore, all the epigenetic modifying factors are regulated by the gdpA promoter derived from Aspergillus nidulans, and the nucleotide sequence of the gdpA promoter is shown in SEQ ID NO.1.

[0010] Furthermore, the preferred species of *Pyctomyces sheathii* is *Pyctomyces sheathii* (…). Coleophoma empetri B9, deposited at the China Center for Type Culture Collection, accession number CCTCC NO.M 20251966, date of deposit: September 3, 2025.

[0011] Furthermore, the overexpression of the epigenetic modifying factors is performed using the vector pDHt, which is introduced into the host bacteria via Agrobacterium-mediated transformation.

[0012] Furthermore, the recombinant genetically engineered bacteria are constructed as follows: (1) the epigenetic modification factor gene and promoter are linked to the vector pDHt to construct a recombinant vector containing the target gene; (2) the recombinant vector containing the target gene is transformed into Agrobacterium; (3) the Agrobacterium from step (2) is co-cultured with the host bacterium *Pteris vittata* spore liquid, and positive transformants that produce high yields of micafungin precursor FR901379 are screened to obtain the recombinant genetically engineered bacteria.

[0013] The present invention also provides the application of the recombinant genetically engineered bacteria in the fermentation preparation of micafungin precursor FR901379.

[0014] Furthermore, the application method is as follows: the recombinant genetically engineered bacteria are inoculated into a fermentation medium and cultured at 20-30℃ and 100-300 rpm (cultured at 25℃ and 250 rpm for 10 days) to obtain a fermentation broth containing the micafungin precursor FR901379; the fermentation medium composition is: glucose 10g / L, sorbitol 100g / L, soybean meal 18g / L, triammonium citrate 3g / L, FeSO4 0.3g / L, (NH4)2SO4 3g / L, MnSO4 0.5g / L, anhydrous MgSO4 2g / L, KH2PO4 1g / L, CaCO3 4g / L, with water as the solvent and normal pH value.

[0015] Furthermore, before fermentation, the recombinant genetically engineered bacteria undergo seed culture expansion, and then the seed culture is inoculated into the fermentation medium at a volume concentration of 10%. The seed culture is obtained by inoculating the engineered bacteria into the seed culture medium and culturing at 25°C and 250 rpm for 2 days. The seed culture medium consists of: 20 g / L glucose, 10 g / L corn starch, 10 g / L soybean meal, 2 g / L KH2PO4, with water as the solvent and a pH of 5.5.

[0016] The present invention also provides a *Pyrodactylus* species for constructing the recombinant genetically engineered bacteria. Coleophoma empetri B9, deposited at the China Center for Type Culture Collection, accession number CCTCC NO.M 20251966, date of deposit: September 3, 2025.

[0017] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0018] This invention provides a recombinant genetically engineered bacterium that produces FR901379 at high yield, containing histone methyltransferase. Dot1 Histone methyltransferase Set2 or histone deacetylase Rpd3 The overexpression vector was introduced into the host strain via Agrobacterium tumefaciens-mediated transformation to obtain the recombinant genetically engineered strain that produces high levels of FR901379. Fermentation verification showed that the engineered strain produced FR901379 at a yield 30-40% higher than the original strain. The method described in this invention is characterized by its simplicity, high transformation efficiency, and good genetic stability. (iv) Description of the attached drawings

[0019] Figure 1 This is a schematic diagram of the original carrier pDHt.

[0020] Figure 2 This is a schematic diagram of the recombinant vectors constructed in Examples 2-4.

[0021] Figure 3 This is a gel electrophoresis image; A represents the linearized carrier backbone pDHt (lane 1) of Example 2 and gdpA Gene amplification product (lane 2); B represents pDHt-gdpA plasmid before and after single enzyme digestion in Example 2; C represents recombinant plasmids constructed in Examples 2-4.

[0022] Figure 4 The image shows the colony PCR validation results of transformants obtained by overexpressing epigenetic modifiers. A represents the overexpression of histone methyltransferases. Dot1 The transformant, B represents overexpression of histone methyltransferase. Set2 The transformant, C represents overexpression of histone deacetylase Rpd3 The transformant, D represents overexpression of histone acetyltransferase Gcn5 Transformants; where M: 250bp DNA marker; 1-8: transformants; WT: originating strain Coleophoma empetri B9.

[0023] Figure 5 Example 2 illustrates the effect of engineered bacteria on the yield of FR901379.

[0024] Figure 6 Example 3 illustrates the effect of engineered bacteria on the yield of FR901379.

[0025] Figure 7 Example 4 illustrates the effect of engineered bacteria on the yield of FR901379.

[0026] Figure 8 Example 5 illustrates the effect of engineered bacteria on the yield of FR901379. (V) Detailed Implementation Methods

[0027] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0028] Aspergillus nidus used in the embodiments of the present invention ( Aspergillus nidulans ZJB09223, with accession number CCTCC NO.M 2012300, has been published in patent application CN103509840A.

[0029] The herbaceous stem spotting used in the embodiments of this invention ( Coleophoma empetri B9, with accession number CCTCCNO.M 20251966. It should be noted that the scope of this invention is not limited to this specific strain.

[0030] The carrier pDHt is commercially available. Unless otherwise specified, all raw materials and equipment used in this invention are commonly used in the field; unless otherwise specified, all methods used in this invention are conventional methods in the field.

[0031] The primer sequence information used in Examples 2-4 is shown in Table 1.

[0032] Table 1 Primer sequence information

[0033]

[0034] In the following examples, the content of FR901379 was determined by HPLC detection, and the specific detection method is as follows:

[0035] The high-performance liquid chromatograph (HPLC) was a Thermo Fisher UltiMate 3000, with a Green Grass C18 column (GH0525046C18A) 5 μm (4.6 mm × 250 mm); a UV detector; a mobile phase of acetonitrile: 0.05 mol / L NaH2PO4 = 50:50 (v / v); a flow rate of 1.0 mL / min; an injection volume of 20 μL; a detection wavelength of 210 nm; and a data acquisition time of 20 min.

[0036] In the following examples, the culture medium composition is as follows:

[0037] LB liquid medium consists of 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride, dissolved in water at a natural pH. LB solid medium is LB liquid medium with 15 g / L agar added.

[0038] The IM liquid induction medium consisted of: 1.8 g / L glucose, 5 mL / L glycerol, 400 mL / L 2.5 × MM salt solution, 40 mL / L 40 mM MES (2-morpholinoethanesulfonic acid) aqueous solution, 1 mL / L 200 μM AS (acetylsyleugenone) aqueous solution, 1.181 mL / L 30% CaCl2 aqueous solution, and 26.875 mL / L 10% MgSO4 aqueous solution. The solvent was water, and the pH was natural. The 2.5 × MM salt solution consisted of: 1.25 g / L ammonium sulfate, 0.375 g / L sodium chloride, 5.125 g / L potassium dihydrogen phosphate, and 3.75 g / L dipotassium hydrogen phosphate. The solvent was water.

[0039] Preparation of IM liquid induction medium (200 mL): 0.36 g glucose, 1 mL glycerol, 112 mL water. Sterilize at 115 °C for 20 minutes. After cooling, add 80 mL of 2.5 × MM salt solution, 8 mL of 40 mM MES, 200 μL of 200 μM AS, 236.2 μL of 30% CaCl2, and 5.375 mL of 10% MgSO4. 2.5 × MM salt solution (500 mL): 0.625 g ammonium sulfate, 0.1875 g sodium chloride, 2.5625 g potassium dihydrogen phosphate, 1.875 g dipotassium hydrogen phosphate, and water to a final volume of 494 mL. Sterilize at 121 °C for 20 minutes.

[0040] The co-culture plate medium is obtained by adding 16 g / L agar powder to IM liquid induction medium and then solidifying it.

[0041] PDA solid culture medium composition: potato 200g / L, glucose 20g / L, agar 15g / L, solvent is water.

[0042] The PDA solid screening medium is composed of 100 μL of 200 μM cefotaxime aqueous solution, 100 μL of 0.1% Triton X-100 aqueous solution, and 100 μL of 50 g / L hygromycin aqueous solution added to 100 mL of PDA solid medium.

[0043] The seed culture medium consisted of 20 g / L glucose, 10 g / L corn starch, 10 g / L soybean meal, and 2 g / L KH2PO4, with water as the solvent. The pH was adjusted to 5.5 with NaOH. The medium was sterilized at 115°C for 30 minutes.

[0044] Fermentation medium composition: glucose 10g / L, sorbitol 100g / L, soybean meal 18g / L, triammonium citrate 3g / L, FeSO4 0.3g / L, (NH4)2SO4 3g / L, MnSO4 0.5g / L, anhydrous MgSO4 2g / L, KH2PO4 1g / L, CaCO3 4g / L, solvent is water, pH value is normal; sterilize at 115℃ for 30 minutes.

[0045] The room temperature mentioned in this invention refers to 25-30℃.

[0046] Example 1: Screening and identification of *Pseudomonas sheathii* B9

[0047] 1. Screening of strain B9

[0048] Using *Coleophoma empetri-F11899* (purchased from BioVector NTCC Type Culture Collection) as the starting strain, the spores were inoculated into PDA liquid medium. In the first round of UV mutagenesis, the spores were irradiated at a time gradient of 30-330 s at room temperature, under a 15 W UV lamp and an irradiation distance of 30 cm. After standing in the dark for 2 h, the spore suspension was spread onto PDA plates inoculated with *Candida albicans* using the Oxford cup method. The plates were then cultured in the dark for 4-5 days to screen for strains with smaller colony morphology but larger inhibition zones against *Candida albicans*. The yield of FR901379 was further screened by shake-flask fermentation as described in step S6 of Example 2, resulting in a mutant strain K7 with increased yield (360 mg / L, compared to 168 mg / L for the original strain). Then, using K7 as the starting strain, a second round of mutagenesis was carried out under the same ultraviolet mutagenesis conditions. After the same screening process, a stable strain B9 with high FR901379 production was finally obtained. Its FR901379 production was stably maintained between 1100-1200 mg / L during the passage.

[0049] 2. Identification of strain B9

[0050] The genome of B9 was extracted using the fungal genome extraction kit from MP Company. After extraction, PCR amplification of the conserved regions of fungal ribosomes was performed using the universal primers ITS1 / ITS4. According to the PCR amplification results observed by the gel imaging system, the target band was found to be between 500 and 800 bp, which is consistent with the size of the ITS sequence band. The subsequent sequencing work was completed by Qingke Biotechnology. The sequencing results of B9 (5-3') are shown below, SEQ ID NO.6.

[0051] .

[0052] The sequencing results were compared with known sequences in the NCBI nucleic acid library to determine that strain B9 belongs to... Coleophoma genus, among which Coleophoma empetri The similarity was 97.50%. Therefore, it was named *Pseudomonas sheathii* (…). Coleophoma empetri B9, deposited at the China Center for Type Culture Collection, accession number CCTCC NO.M20251966, date of deposit: September 3, 2025.

[0053] Example 2: Construction, fermentation, and screening of the engineered strain pDHt-gdpA-Dot1 of *Pittosporum tobira*.

[0054] S1. Construct the recombinant vector pDHt-gdpA-Dot1

[0055] The structural diagram of the carrier pDHt is shown below. Figure 1 As shown, the linearized vector backbone pDHt was obtained using primers 5UTR-F / Trpc-R. The gel electrophoresis image is shown below. Figure 3 Lane 1 of the middle A swimming lane.

[0056] Using the genome of Aspergillus nidulans CCTCC NO.M 2012300 as a template, primer pairs gdpA-F / gdpA-R (Table 1) were used to amplify... gdpA The promoter gene fragment (nucleotide sequence as shown in SEQ ID NO.1), the PCR amplification system is shown in Table 2, the PCR amplification program is shown in Table 3, and the gel electrophoresis image of the amplification product is shown in Table 3. Figure 3 Lane 2 of the middle A section.

[0057] Ligation was performed using the non-ligase-dependent single-fragment rapid cloning kit (ClonExpress II One Step Cloning Kit, catalog number C112, Vazyme). gdpA The promoter gene fragment and linearized pDHt were sequenced and verified to obtain the recombinant plasmid pDHt-gdpA.

[0058] Using the genome of *Pseudomonas sheathiformis* B9 as a template, the histone methyltransferase Dot1 was amplified using primer pair Dot1-F / Dot-R (Table 1). Dot1 Fragment (nucleotide sequence as shown in SEQ ID NO.2), pDHt-gdpA plasmid used SpeI Single enzyme digestion was performed. Gel electrophoresis images of the digestion products and the pDHt-gdpA plasmid before digestion are shown below. Figure 3 Lanes 1 and 2 of swimmer B; then connect using the ClonExpress II One Step Cloning Kit. Dot1 Genes and pDHt-gdpA ( SpeI Sequencing confirmed the recombinant plasmid pDHt-gdpA-Dot1. The plasmid structure is shown below. Figure 2 See gel electrophoresis image for A. Figure 3 Lane 1 of the middle C swimming lane.

[0059] Table 2. PCR amplification system

[0060]

[0061] Table 3. PCR Amplification Procedure

[0062]

[0063] S2. Activation and scale-up culture of Agrobacterium containing pDHt-gdpA-Dot1 plasmid

[0064] The vector pDHt-gdpA-Dot1 was introduced into Agrobacterium tumefaciens via chemical transformation. Agrobacterium tumefaciensA *Agrobacterium* strain carrying the vector pDHt-gdpA-Dot1 was obtained from the genome of AGL1 competent cells. The positive *Agrobacterium* strain was inoculated onto LB solid medium and cultured at 28°C for 60 h. The positive *Agrobacterium* strain was then picked and cultured in LB liquid medium at 28°C with shaking at 200 rpm for 48 h. The culture medium was centrifuged at 4000 rpm for 5 min. The collected bacterial pellet was resuspended in IM liquid induction medium to an absorbance value of 600 nm. 600 The concentration was set to 0.3, and then the mixture was incubated at 28°C with shaking for 6 hours to obtain the OD value. 600 Agrobacterium bacterial suspension with a value of 1;

[0065] S3. Preparation of suspension of fungal spores from the sheath stem

[0066] *Pseudomonas sheathii* B9 was inoculated onto PDA solid medium plates and activated at 25°C for 10 days until spores were produced. The mycelial surface was scraped with physiological saline, and the mycelium was filtered through three layers of lens paper. The concentration was adjusted with physiological saline to obtain a spore concentration of 10. 6 A suspension of fungal spores per ml from the stem sheath;

[0067] S4, co-culture of Agrobacterium and Pyrodactylus sheathii

[0068] The OD obtained in step S2 600 Agrobacterium bacterial suspension of 1 and 10 obtained in step S3 6 The suspension of *Pseudomonas aeruginosa* spores per ml was mixed in equal volumes to obtain a mixture; the mixture was then evenly spread on a co-culture plate culture medium pre-lined with sterile cellophane and incubated in the dark at 25°C for 48 hours to obtain a co-culture.

[0069] S5. Initial screening and identification of transformants

[0070] Transfer the cellophane and co-culture from step S4 to an empty plate. Cover the cellophane and co-culture with PDA solid selection medium and incubate at 25°C in the dark for 7-10 days until transformants emerge. Extract the genome from the emerged *Pittosporum tobira* transformants and perform colony PCR verification using the conditions shown in Tables 4 and 5. Figure 4 (A) The strain that is correctly verified by PCR is the primary screening transformant of *Tectus sheathii* whose genome contains *Agrobacterium* T-DNA.

[0071] The primary screening transformants of *Pyrhodotorula buergerianum* were selected and inoculated onto a separate PDA solid selection medium. The medium was cultured at 25°C until *Pyrhodotorula buergerianum* strains grew on the surface of the PDA solid selection medium. The grown *Pyrhodotorula buergerianum* strains were the stably growing *Pyrhodotorula buergerianum* transformants with pDHt-gdpA-Dot1 inserted into their genome. The engineered *Pyrhodotorula buergerianum* strains containing the pDHt-gdpA-Dot1 plasmid were screened and designated as strains OEDot1-1, OEDot1-2, OEDot1-3, OEDot1-4, and OEDot1-5.

[0072] Table 4. Composition of the Colony Validation PCR System

[0073]

[0074] Table 5. Colony Validation PCR Procedure

[0075]

[0076] S6, Fermentation and Screening of Engineered Strains

[0077] Strains of *Pseudomonas sheathii* B9 (control), OEDot1-1, OEDot1-2, OEDot1-3, OEDot1-4, and OEDot1-5 were inoculated into 50 mL of seed culture medium and cultured at 25℃ and 250 rpm for 2 days. Then, 10% (v / v) of the inoculum was added to 50 mL of fermentation culture medium and cultured at 25℃ and 250 rpm for 10 days. 1 mL of fermentation broth was mixed thoroughly with 1 mL of pure methanol, sonicated for 60 min, filtered, and the filtrate was analyzed using high-performance liquid chromatography (HPLC) to determine the yield of FR901379. The filter cake was dried at 80℃ until its weight remained constant, and the dry weight (DCW) of the bacterial cells was measured.

[0078] The results of shake-flask fermentation are as follows Figure 5 As shown, the FR901379 yields of strains OEDot1-2 and OEDot1-5 were higher than those of the control strain, at 1.47 g / L and 1.50 g / L, respectively, which were 37.4% and 40.1% higher than the yield of the control strain (1.07 g / L).

[0079] Example 3: Construction, fermentation, and screening of the engineered strain pDHt-gdpA-Set2 of *Pittosporum tobira*.

[0080] Using the genome of *Pseudomonas sheathii* B9 as a template, the histone methyltransferase Set2 was amplified using primer pair Set2-F / Set2-R (Table 1). Set2 Gene fragment (nucleotide sequence as shown in SEQ ID NO.3).

[0081] Using the same methods and conditions as in Example 2, the recombinant plasmid pDHt-gdpA-Set2 was constructed sequentially (see gel electrophoresis image). Figure 3 Lane 2 of C), introduced into Agrobacterium AGL1, activated and expanded culture, co-culture, initial screening and identification of transformants ( Figure 4 (B) Screening yielded *Pteris vittata* containing the pDHt-gdpA-Set2 plasmid, which were named strains OESet2-1, OESet2-2, OESet2-3, OESet2-4, and OESet2-5.

[0082] Using the same methods and conditions as in Example 2, *Pseudomonas sheathii* B9 (control), strains OESet2-1, OESet2-2, OESet2-3, OESet2-4, and OESet2-5 were subjected to shake-flask fermentation. The results are as follows: Figure 6 As shown, the FR901379 yield of strain OESet2-5 was higher than that of the control strain, at 1.42 g / L, which was 33% higher than that of the control strain (1.07 g / L).

[0083] Example 4: Construction, fermentation, and screening of the engineered strain pDHt-gdpA-Rpd3 of *Pittosporum tobira*.

[0084] Using the genome of *Pseudomonas sheathii* B9 as a template, the histone deacetylase Rpd3 was amplified using primer pair Rpd3-F / Rpd3-R (Table 1). Rpd3 Gene fragment (nucleotide sequence as shown in SEQ ID NO.4).

[0085] The recombinant plasmid pDHt-gdpA-Rpd3 was constructed sequentially using the same methods and conditions as in Example 2 (see gel electrophoresis image). Figure 3 Lane 3 of the middle C section), introduced into Agrobacterium AGL1, activated and expanded culture, co-culture, initial screening and identification of transformants ( Figure 4 (C) Screening yielded *Pyrhodops stenoptera* containing the pDHt-gdpA-Rpd3 plasmid, which were designated as strains OERpd3-1, OERpd3-2, OERpd3-3, OERpd3-4, and OERpd3-5.

[0086] Using the same methods and conditions as in Example 2, *Pseudomonas sheathii* B9 (control), strains OERpd3-1, OERpd3-2, OERpd3-3, OERpd3-4, and OERpd3-5 were subjected to shake-flask fermentation. The results are as follows: Figure 7 As shown, the FR901379 yield of strain OERpd3-1 was higher than that of the control strain, at 1.49 g / L, which was 39% higher than that of the control strain (1.07 g / L).

[0087] Example 5: Construction, fermentation, and screening of the engineered strain pDHt-gdpA-Gcn5 of *Pittosporum tobira*.

[0088] Using the genome of *Pseudomonas sheathiformis* B9 as a template, the histone acetyltransferase Gcn5 was amplified using primer pair Gcn5-F / Gcn5-R (Table 1). Gcn5 Gene fragment (nucleotide sequence as shown in SEQ ID NO.5).

[0089] The recombinant plasmid pDHt-gdpA-Gcn5 was constructed sequentially using the same methods and conditions as in Example 2 (see gel electrophoresis image). Figure 3 Lane 4 of the middle C section), introduced into Agrobacterium AGL1, activated and expanded culture, co-culture, initial screening and identification of transformants ( Figure 4 (D) Screening yielded *Pyrhodops stenoptera* containing the pDHt-gdpA-Gcn5 plasmid, which were designated as strains OEGcn5-1, OEGcn5-2, OEGcn5-3, OEGcn5-4, and OEGcn5-5.

[0090] Using the same methods and conditions as in Example 2, *Pseudomonas sheathii* B9 (control), strains OEGcn5-1, OEGcn5-2, OEGcn5-3, OEGcn5-4, and OEGcn5-5 were subjected to shake-flask fermentation. The results are as follows: Figure 8 As shown, the FR901379 yields of strains OEGcn5-1, OEGcn5-2, OEGcn5-3, OEGcn5-4, and OEGcn5-5 were all lower than those of the control strain.

[0091] While the invention has been specifically described in conjunction with various preferred embodiments, it should be understood that the invention is not limited to these specific embodiments. In fact, various modifications that will be obvious to those skilled in the art as described above to obtain the invention should be included within the scope of this invention.

Claims

1. A recombinant genetically engineered bacterium producing micafungin precursor FR901379, characterized in that, The recombinant genetically engineered bacteria were obtained from the fungus *Pittosporum tobira* (…). Coleophoma empetri Recombinant genetically engineered bacteria that overexpressed epigenetic modifiers in their genome and screened for high-yield micafungin precursor FR901379 were obtained; the epigenetic modifiers included histone methyltransferases. Dot1 Histone methyltransferase Set2 or histone deacetylase Rpd3 .

2. The recombinant genetically engineered bacteria as described in claim 1, characterized in that, The histone methyltransferase Dot1 The nucleotide sequence is shown in SEQ ID NO.2; the histone methyltransferase Set2 The nucleotide sequence is shown in SEQ ID NO.3; the histone deacetylase Rpd3 The nucleotide sequence is shown in SEQ ID NO.

4.

3. The recombinant genetically engineered bacteria as described in claim 1, characterized in that, The epigenetic modifying factors are all regulated by the gdpA promoter derived from Aspergillus nidulans, and the nucleotide sequence of the gdpA promoter is shown in SEQ ID NO.

1.

4. The recombinant genetically engineered bacteria as described in claim 1, characterized in that, The *Pycium sheathii* is *Pycium sheathii* ( Coleophoma empetri B9, deposited at the China Center for Type Culture Collection, accession number CCTCC NO.M20251966, date of deposit: September 3, 2025.

5. The recombinant genetically engineered bacteria as described in claim 1, characterized in that, The overexpression of the epigenetic modifiers was performed using the vector pDHt, which was introduced into the host bacteria via Agrobacterium-mediated transformation.

6. The recombinant genetically engineered bacteria according to any one of claims 1-5, characterized in that, The recombinant genetically engineered bacteria were constructed as follows: (1) the epigenetic modification factor gene and promoter were linked to the vector pDHt to construct a recombinant vector containing the target gene; (2) the recombinant vector containing the target gene was transformed into Agrobacterium; (3) the Agrobacterium from step (2) was co-cultured with the host bacterium *Pteris vittata* spore liquid, and positive transformants producing high-yield micafungin precursor FR901379 were screened to obtain the recombinant genetically engineered bacteria.

7. The use of the recombinant genetically engineered bacteria of claim 1 in the fermentation preparation of micafungin precursor FR901379.

8. The application as described in claim 7, characterized in that, The application method is as follows: the recombinant genetically engineered bacteria are inoculated into a fermentation medium and cultured at 20-30℃ and 100-300rpm to obtain a fermentation broth containing the micafungin precursor FR901379; the fermentation medium composition is: glucose 10g / L, sorbitol 100g / L, soybean meal 18g / L, triammonium citrate 3g / L, FeSO4 0.3g / L, (NH4)2SO4 3g / L, MnSO4 0.5g / L, anhydrous MgSO4 2g / L, KH2PO4 1g / L, CaCO3 4g / L, with water as the solvent and natural pH value.

9. The application as described in claim 8, characterized in that, Before fermentation, the recombinant genetically engineered bacteria undergo seed culture expansion. Then, the seed culture is inoculated into the fermentation medium at a volume concentration of 10%. The seed culture is obtained by inoculating the engineered bacteria into the seed culture medium and culturing at 25°C and 250 rpm for 2 days. The seed culture medium consists of: 20 g / L glucose, 10 g / L corn starch, 10 g / L soybean meal, 2 g / L KH2PO4, water as solvent, and pH 5.

5.

10. A method for constructing the recombinant genetically engineered bacterium of claim 1, *Pyrodactylus* (a type of fungus). Coleophoma empetri B9, deposited at the China Center for Type Culture Collection, accession number CCTCC NO.M 20251966, date of deposit: September 3, 2025.

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