Phialophora engineering bacteria for efficient production of fr901379, construction method and application thereof

Abstract

The application discloses a high-yield FR901379 Sarcodina stem point mold engineering bacteria and a construction method and application. McfG , McfM and McfN are introduced into the Sarcodina stem point mold to achieve the purpose of integration into the genome, so that the Sarcodina stem point mold genetic engineering bacteria strain capable of stable growth and greatly improved FR901379 yield is obtained, and the yield can be increased by 32.65% at most compared with the original strain. Meanwhile, under the same fermentation medium culture conditions, the strain constructed by the application can obviously shorten the fermentation period, reduce energy consumption, bacterial contamination risk and production cost, and help the industrialized efficient production. In addition, the method has the characteristics of simple operation, high efficiency, high accuracy and the like.

Description

(I) Technical Field

[0002] This invention belongs to the field of microbial and genetic engineering technology, and relates to an engineered fungus for the efficient production of FR901379, its construction method, and its application. (II) Background Technology

[0004] Micafungin, a novel antifungal drug, has its prodrug FR901379, a novel cyclic lipopeptide compound. Belonging to the echinocandin class of drugs, its mechanism of action primarily involves specifically targeting the synthesis of 1,3-β-D-glucan on the fungal cell membrane, irreversibly blocking the cell wall synthesis pathway and thus killing the fungal cells. Currently, micafungin has a much wider range of clinical applications than caspofungin and anidulafungin due to its superior pharmacokinetic properties, broader antifungal spectrum, and higher safety profile.

[0005] Existing research indicates that the synthetic substrate microbial spectrum of micafungin precursor FR901379 is narrow, with *Pyctomyces sheathii* (…). Coleophoma empetri The only naturally occurring strain of *Pyrrosia lingua* is the core strain for the synthesis of this precursor. However, existing methods for synthesizing the micafungin precursor FR901379 using *Pyrrosia lingua* suffer from four main drawbacks: low yield, difficult genetic manipulation, high fermentation cost, and numerous byproducts. These directly restrict industrial scale-up and economic viability. First, the fermentation yield of wild-type *Pyrrosia lingua* FR901379 is far lower than the requirements for industrial production. The fermentation process suffers from viscous fermentation broth, long fermentation cycles, and susceptibility to contamination, becoming the core bottleneck limiting FR901379 yield improvement. Second, there are deficiencies in genetic modification and molecular tools. Traditional protoplast transformation methods are inefficient, and the lack of efficient screening markers results in extremely low positive transformant yields. Furthermore, the presence of byproducts such as the WF11899 complex and FR179642 during fermentation leads to high isolation costs and low purity of the target product FR901379. (III) Summary of the Invention

[0007] The purpose of this invention is to provide an engineered strain of *Pyrtomyces luteoliaceus* for the efficient production of FR901379, its construction method, and its application. By enhancing the activity of key catalytic enzymes in the metabolic synthesis process of FR901379, the fermentation yield of the target product can be increased and the fermentation cycle can be shortened.

[0008] The technical solution adopted in this invention is:

[0009] This invention provides an engineered strain of *Pyrophyllus sheathii* that efficiently produces FR901379. The engineered strain is *Pyrophyllus sheathii* strain that overexpresses the 3S-hydroxy-L-high tyrosine synthesis gene in the *Pyrophyllus sheathii* genome. McfG ), ATP-binding cassette (ABC) transporter synthesis gene ( McfM) Or 3-hydroxy-L-glutamine synthesis gene ( McfN) One of the selected strains was *Pittosporum tobira*, which showed a significant increase in yield.

[0010] Preferably, the McfG The nucleotide sequence is shown in SEQ ID NO.2; McfM The nucleotide sequence is shown in SEQ ID NO. 3; McfN The nucleotide sequence is shown in SEQ ID NO.4.

[0011] Preferably, the McfG , McfM as well as McfN All were regulated by the gpdA promoter derived from Aspergillus nidulans, and the nucleotide sequence of the gpdA promoter is shown in SEQ ID NO.1.

[0012] Preferably, the engineered fungus *Pyrhoa sheathis* is constructed as follows: (1) the target gene and promoter are ligated to the restriction endonuclease of the vector pDHt. SpeI and XbaI (1) Construct a vector containing the target gene between the loci; (2) Transform the vector containing the target gene into Agrobacterium using Agrobacterium transformation method and prepare Agrobacterium bacterial solution; (3) Mix the Agrobacterium bacterial solution from step (2) with the suspension of Pterygotic spores, spread them on IM plates for co-culture, screen positive transformants with a significant increase in FR901379 yield, and obtain the engineered Pterygotic spore strain.

[0013] Preferably, the Agrobacterium in step (2) is Agrobacterium AGL1.

[0014] Preferably, the method for preparing Agrobacterium culture in step (2) is as follows: Inoculate positive Agrobacterium strains onto LB solid medium and culture at 28°C for 60 h; pick colonies and culture them in LB liquid medium at 28°C with shaking at 200 rpm for 48 h; centrifuge the culture medium at 4000 rpm for 5 min; resuspend the collected bacterial pellet in IM liquid induction medium to OD. 600 The concentration was set to 0.3, and then the mixture was incubated at 28°C with shaking for 6 hours until the OD value reached 0.3. 600The culture medium was 1, and the expanded Agrobacterium tumefaciens culture was obtained. The IM liquid induction medium consisted of: 5 mL / L glycerol, 1 mL / L 200 μM AS (acetylsyleugenone) aqueous solution, 1.8 g / L glucose, 1.181 mL / L 30% CaCl2 aqueous solution, 400 mL / L 2.5×MM salt solution, 26.875 mL / L 10% MgSO4 aqueous solution, and 40 mL / L 40 mM MES (2-morpholinoethanesulfonic acid) aqueous solution, with natural pH. The 2.5×MM salt solution consisted of: 1.25 g / L (NH4)2SO4, 0.375 g / L NaCl, 5.125 g / L KH2PO4, and 3.75 g / L K2HPO4, with water as the solvent. The IM plate medium was prepared by adding 16 g / L agar powder to the IM liquid induction medium.

[0015] Preferably, the method for preparing the *Pyrtomyces sheathii* spore suspension in step (3) is as follows: *Pyrtomyces sheathii* is inoculated onto a PDA plate medium and activated at 25°C for 10 days until mature spores are produced. After that, the colony surface is scraped with sterile physiological saline. The resulting mixture of mycelia and physiological saline is filtered through three layers of lens paper to obtain a fresh spore suspension. The concentration is then adjusted to 10% with physiological saline. 6 per mL.

[0016] Preferably, in step (3), the OD of Agrobacterium bacterial solution 600 The concentration of the fungal suspension in the sheath stem was 1, and the concentration of the fungal suspension was 10. 6 The number of cells / mL was 1:1.

[0017] This invention provides a method for the efficient fermentation production of FR901379 using the engineered strain *Pyrtomyces sheathii*. The method comprises: inoculating the engineered strain into a seed culture medium and culturing it at 25°C and 240 rpm for 2 days to obtain a seed solution; then inoculating the seed solution into a fermentation medium at a volume concentration of 10%; and culturing it on a shaker at 25°C and 240 rpm for 10 days to obtain a fermentation broth containing FR901379. 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 pH adjusted to 5.5 using NaOH. The fermentation medium consists of: 10 g / L glucose, 100 g / L sorbitol, 18 g / L soybean meal, 3 g / L triammonium citrate, 3 g / L (NH₄)₂SO₄, 1 g / L KH₂PO₄, and 2 g / L MgSO₄. The concentrations of the following components are present: g / L MnSO4 0.5 g / L, FeSO4 0.3 g / L, CaCO3 4 g / L, with water as the solvent and normal pH value.

[0018] Compared with the prior art, the beneficial effects of this invention are mainly reflected in: this invention introduces the 3S-hydroxy-L-high tyrosine synthesis gene ( McfG ), ATP-binding cassette (ABC) transporter synthesis gene ( McfM ) and the 3-hydroxy-L-glutamine synthesis gene ( McfN One of the methods is integrated into an expression vector, and then transformed into the genome of *Agrobacterium sheathii* using Agrobacterium-mediated transformation (AMT), thereby obtaining a *Agrobacterium sheathii* genetically engineered strain that can grow stably and significantly increase the yield of FR901379. The *Agrobacterium sheathii* genetically engineered strain constructed in this invention can increase the yield of FR901379 by up to 32.6% compared to the original strain. Furthermore, under the same fermentation medium and culture conditions, the strain constructed in this invention achieves nearly the same yield in a shorter time compared to the high-yielding strain constructed in patent CN117568378A, significantly shortening the fermentation cycle, reducing energy consumption, risk of contamination, and production costs, thus contributing to efficient industrial production. In addition, the method of this invention is simple and efficient to operate, and also has high accuracy. (iv) Description of the attached drawings

[0020] Figure 1 This is a schematic diagram of the original carrier pDHt.

[0021] Figure 2 This is a schematic diagram of the structure of the carrier pDHt-gpdA-McfG.

[0022] Figure 3 This is a schematic diagram of the structure of the carrier pDHt-gpdA-McfM.

[0023] Figure 4 This is a schematic diagram of the structure of the carrier pDHt-gpdA-McfN.

[0024] Figure 5 As a carrier chromatin regulator CclA A structural diagram.

[0025] Figure 6 Gene for the synthesis of 3S-hydroxy-L-high tyrosine McfG The effect of overexpression on FR901379 yield.

[0026] Figure 7 Gene for the synthesis of ATP-binding cassette (ABC) transporter proteins McfM The effect of overexpression on FR901379 yield.

[0027] Figure 8 Gene for the synthesis of 3-hydroxy-L-glutamine McfN The effect of overexpression on FR901379 yield.

[0028] Figure 9 Chromatin regulator CclA The effect of overexpression on FR901379 yield.

[0029] Figure 10 Fermentation of control strain (a) and MG5 strain (b) under the same culture conditions. (V) Detailed Implementation Methods

[0031] 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:

[0032] This invention uses HPLC to detect the yield of FR901379. The specific detection method is as follows:

[0033] The high-performance liquid chromatograph (HPLC) was a Thermo Fisher UltiMate 3000, with a Green Grass C18 column (GH0525046C18A, 4.6 mm × 250 m, 5 μm); a UV detector; a mobile phase of acetonitrile: 0.05 mol / L NaH2PO4 aqueous solution = 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 peak elution time of FR901379 of 20 min.

[0034] The culture medium used in this invention has the following composition:

[0035] LB liquid medium: yeast extract 5 g / L, sodium chloride 10 g / L, tryptone 10 g / L, solvent: water, pH: natural. LB solid medium is LB liquid medium with 15 g / L agar added.

[0036] The IM liquid induction medium consisted of: 5 mL / L glycerol, 1 mL / L 200 μM acetobutanone aqueous solution, 1.8 g / L glucose, 1.181 mL / L 30% CaCl2 aqueous solution, 400 mL / L 2.5×MM salt solution, 26.875 mL / L 10% MgSO4 aqueous solution, and 40 mL / L 40 mM 2-morpholine ethanesulfonic acid aqueous solution, with natural pH. The 2.5×MM salt solution consisted of: 1.25 g / L (NH4)2SO4, 0.375 g / L NaCl, 5.125 g / L KH2PO4, and 3.75 g / L K2HPO4, with water as the solvent.

[0037] Preparation method of IM liquid induction medium: 0.36 g glucose, 1 mL glycerol, 112 mL H2O, sterilize at 115 ℃ for 20 minutes. After cooling, add 80 mL of 2.5×MM salt solution, 8 mL of 40 mM MES aqueous solution, 200 μL of 200 μM AS aqueous solution, 236.2 μL of 30% CaCl2 aqueous solution, and 5.375 mL of 10% MgSO4 aqueous solution. 2.5×MM salt solution (500 mL): 0.625 g (NH4)2SO4, 0.1875 g NaCl, 2.5625 g KH2PO4, 1.875 g K2HPO4, add water to a final volume of 494 mL, sterilize at 121 ℃ for 20 minutes.

[0038] Co-culture IM plate medium: obtained by adding 16 g / L agar powder to IM liquid induction medium and heating to solidify.

[0039] PDA solid culture medium composition: glucose 20 g / L, peeled potato 200 g / L, agar 15 g / L, solvent is water.

[0040] The PDA solid screening medium consists of 100 μL of 200 μM cefotaxime aqueous solution, 100 μL of 50 g / L hygromycin aqueous solution, and 100 μL of 0.1% Triton X-100 aqueous solution.

[0041] The seed culture medium consisted of 20 g / L glucose, 10 g / L soybean meal, 2 g / L KH2PO4, 10 g / L corn starch, and water as the solvent. The pH was adjusted to 5.5 using NaOH. The medium was sterilized at 115 °C for 30 minutes.

[0042] Fermentation medium composition: glucose 10 g / L, sorbitol 100 g / L, soybean meal 18 g / L, triammonium citrate 3 g / L, FeSO4 0.3 g / L, (NH4)2SO4 3 g / L, MnSO4 0.5 g / L, anhydrous MgSO4 2 g / L, KH2PO4 1 g / L, CaCO3 4 g / L, solvent: water, pH value: normal; sterilized at 115 ℃ for 30 minutes.

[0043] Aspergillus nidus used in the embodiments of the present invention ( Aspergillus nidulans ZJB09223, accession number CCTCC NO.M2012300, has been disclosed in patent application CN103555591A.

[0044] The herbaceous stem spotting used in the embodiments of this invention ( Coleophoma empetriB9, with accession number CCTCCNO.M 20251966, has been disclosed in patent application CN121320115A. It should be noted that the scope of this invention is not limited to this specific strain.

[0045] The pDHt carrier is commercially available.

[0046] Example 1: Construction, fermentation, and HPLC detection of engineered strains of *Pittosporum tobira*.

[0047] 1. Construction of vector pDHt-gpdA-McfG

[0048] The structural diagram of the carrier pDHt is shown below. Figure 1 As shown, using restriction endonucleases SpeI and XbaI The linearized vector backbone of pDHt was obtained after double enzyme digestion.

[0049] Using the genome of Aspergillus nidulans CCTCC NO.M2012300 as a template, the promoter was amplified using primer pair gpdA-F / gpdA-R (Table 1). gpdA The fragment (nucleotide sequence as shown in SEQ ID NO.1) was then ligated using Vazyme's C115 ligase to connect the target gene. gpdA The fragment was ligated into the linearized vector backbone pDHt (see plasmid map). Figure 1 After sequencing verification confirmed the accuracy of the sequencing, the vector pDHt-gpdA was obtained. Restriction endonuclease was then used again... XbaI The vector pDHt-gpdA was digested with a single enzyme to obtain a linearized vector backbone.

[0050] Using the genome of *Pseudomonas sheathii* B9 as a template, primer pair McfG-F / McfG-R (Table 1) was used to amplify... McfG The fragment (GenBank accession number XM_629004, nucleotide sequence as shown in SEQ ID NO.2) was then used again with the C115 ligase described above to ligate the target gene. McfG The fragment was ligated into the linearized vector backbone pDHt-gpdA. After sequencing verification confirmed the results were correct, the vector pDHt-gpdA-McfG was obtained (see diagram). Figure 2 As shown, the T-DNA region carries the target gene gpdA-McfG.

[0051] Table 1 Primer sequences (-F indicates upstream primer, -R indicates downstream primer)

[0052]

[0053] 2. Construction of engineered strains of *Pyrodactylus sheathiformis* containing the pDHt-gpdA-McfG plasmid using the AMT method.

[0054] A. Activation and scale-up culture of Agrobacterium

[0055] The vector pDHt-gpdA-McfG was introduced into AGL1 Agrobacterium competent cells using chemical transformation to obtain positive Agrobacterium strains carrying the vector. These positive Agrobacterium strains were inoculated onto LB solid medium and cultured at 28°C for 60 h. Colonies were then picked and cultured in LB liquid medium at 28°C with shaking for 48 h. The culture was then centrifuged at 4000 rpm for 5 min. The collected bacterial pellet was resuspended in IM liquid induction medium until the absorbance at 600 nm was 0.3, and then cultured at 28°C with shaking for 6 h to obtain the expanded Agrobacterium culture solution. 600 The value is 1.

[0056] B. Preparation of suspension of fungal spores from sheath stems

[0057] *Pseudomonas sheathii* B9 was inoculated onto PDA plates and activated at 25°C for 10 days until mature spores were produced. The colony surface was then scraped with sterile physiological saline. The resulting mixture of hyphae and physiological saline was filtered through three layers of lens paper to obtain a fresh spore suspension. The concentration was adjusted with physiological saline until the number of spores under microscopic examination was 10. 6 When the count reaches 100 cells / mL, the next step can be performed.

[0058] C. Co-culture of Agrobacterium and Pyrodactylus sheathii

[0059] Take 100 μL of the activated Agrobacterium tumefaciens solution obtained in step A and the fresh Pseudomonas aeruginosa spore suspension obtained in step B and mix them in equal volumes. Then spread the mixture evenly on a co-culture IM plate medium lined with sterile cellophane and incubate at 25°C in the dark for 48 h to obtain the co-culture.

[0060] D. Initial screening and stabilization of transformants

[0061] Transfer the cellophane covered with the co-culture product obtained in step C to a sterile empty glass plate. Take PDA solid screening medium, heat and cool it to a suitable temperature, and then cover it on the cellophane and co-culture. Incubate at 25°C in the dark for 7-10 days. After the transformants grow, extract the genome for colony PCR verification. The strain that is verified correctly is the initial screening transformant of *Pyrrosia lingua* with the pDHt-gpdA-McfG plasmid inserted in its genome.

[0062] Select the correct *Pyrhodotorula buergerianum* primary screening transformants and re-inoculate them onto a separate PDA solid selection medium. Incubate at 25°C until strains grow on the surface of the medium. These are stable *Pyrhodotorula buergerianum* transformants with the pDHt-gpdA-McfG plasmid inserted into their genome. Select the *Pyrhodotorula buergerianum* engineered strains containing the pDHt-gpdA-McfG plasmid and designate them as strains MG1, MG2, MG3, MG4, and MG5.

[0063] 3. Fermentation and screening of engineered strains

[0064] *Pseudomonas sheathii* B9 (control), strains MG1, MG2, MG3, MG4, and MG5 were inoculated into 50 mL of seed culture medium and cultured at 25℃ and 240 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 240 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 used to determine the yield of FR901379 using high-performance liquid chromatography (HPLC).

[0065] The results of shake-flask fermentation are as follows Figure 6 As shown, the FR901379 yield of strain MG5 was higher than that of the control strain, reaching 1666.97 mg / L, which is 32.65% higher than that of the control strain (1256.65 mg / L).

[0066] Example 2: Construction, fermentation, and HPLC detection of an engineered strain of *Pyrodactylus sheathiformis* containing plasmid pDHt-gpdA-McfM.

[0067] 1. Construction of plasmid pDHt-gpdA-McfM

[0068] Using the genome of *Pseudomonas sheathii* B9 as a template, primer pair McfM-F / McfM-R (Table 1) was used to amplify... McfM The fragment (GenBank accession number XM_633899, nucleotide sequence as shown in SEQ ID NO.3) was then ligated using Vazyme's C115 ligase. McfM The linearized vector backbone pDHt-gpdA prepared by the method in Example 1 was sequenced for verification, yielding the vector pDHt-gpdA-McfM (see diagram). Figure 3 (As shown).

[0069] 2. Construction of engineered strains of *Pyroderma sheathii* containing plasmid pDHt-gpdA-McfM

[0070] Using the AMT method described in Example 1, the plasmid pDHt-gpdA-McfM was introduced into *Pyrtomyces sheathi* strain B9, allowing homologous recombination to occur. Engineered strains of *Pyrtomyces sheathi* containing the plasmid pDHt-gpdA-McfM were then screened. The transformants carrying this plasmid were designated MM1, MM2, MM3, MM4, and MM5.

[0071] 3. Fermentation and screening of engineered strain pDHt-gpdA-McfM

[0072] The yield of FR901379 in the control strain *Pyrtomyces sheathii* B9 and transformants MM1, MM2, MM3, MM4, and MM5 was detected using the method described in Example 1. The results of shake-flask fermentation are shown in Figure 7. The FR901379 yield of strain MM5 was higher than that of the control strain, with a yield of 1465.77 mg / L, which is 16.64% higher than that of the control strain (1256.64 mg / L).

[0073] Example 3: Construction, fermentation, and HPLC detection of an engineered strain of *Pittosporum tobira* containing plasmid pDHt-gpdA-McfN.

[0074] 1. Construction of plasmid pDHt-gpdA-McfN

[0075] Using the genome of *Pseudomonas sheathinarum* B9 as a template, primer pair McfN-F / McfN-R (Table 1) was used to amplify... McfN The fragment (GenBank accession number XM_629071, nucleotide sequence as shown in SEQ ID NO.4) was then ligated using Vazyme's C115 ligase. McfN The linearized vector backbone pDHt-gpdA prepared by the method in Example 1 was sequenced for verification, yielding the vector pDHt-gpdA-McfN (see diagram). Figure 4 (As shown).

[0076] 2. Construction of engineered strains of *Pittosporum tobira* containing plasmid pDHt-gpdA-McfN

[0077] Using the AMT method described in Example 1, the plasmid pDHt-gpdA-McfN was introduced into *Pyrtomyces sheathii* strain B9, allowing homologous recombination to occur. Engineered strains of *Pyrtomyces sheathii* containing the plasmid pDHt-gpdA-McfN were then screened. The transformants carrying this plasmid were designated MN1, MN2, MN3, MN4, and MN5.

[0078] 3. Fermentation and screening of engineered strain pDHt-gpdA-McfN

[0079] The yield of FR901379 in control strains *Pyrtomyces sheathii* B9 and strains MN1, MN2, MN3, MN4, and MN5 was detected using the method described in Example 1. The shake-flask fermentation results are as follows: Figure 8 As shown, the FR901379 yield of strain MN1 was significantly higher than that of the control strain, with its yield (1332.17 mg / L) increasing by 10.90% compared to the control strain's yield (1201.22 mg / L).

[0080] Example 4: Construction, fermentation, and HPLC detection of an engineered strain of *Pyrodactylus sheathiformis* containing plasmid pDHt-gpdA-CclA.

[0081] 1. Construction of plasmid pDHt-gpdA-CclA

[0082] Using the genome of *Pseudomonas sheathii* B9 as a template, primers CclA-F / CclA-R (Table 1) were used to amplify... CclA The fragment (GenBank accession number XM_743637, nucleotide sequence as shown in SEQ ID NO.5) was then ligated using Vazyme's C115 ligase. CclA The linearized vector backbone pDHt-gpdA prepared by the method in Example 1 was sequenced for verification, yielding the vector pDHt-gpdA-CclA (see diagram). Figure 5 (As shown).

[0083] 2. Construction of engineered strains of *Pyroderma sheathii* containing plasmid pDHt-gpdA-CclA

[0084] Using the AMT method described in Example 1, the plasmid pDHt-gpdA-CclA was introduced into *Pyrrosia lingua* strain B9, inducing homologous recombination. Engineered strains of *Pyrrosia lingua* containing the plasmid pDHt-gpdA-CclA were then screened. The transformants carrying this plasmid were designated CclA-1, CclA-2, CclA-3, and CclA-4.

[0085] 3. Fermentation and screening of engineered strain pDHt-gpdA-CclA

[0086] The yield of FR901379 in control *Pyctomyces sheathii* B9 and strains CclA-1, CclA-2, CclA-3, and CclA-4 was detected using the method described in Example 1. The shake-flask fermentation results are as follows: Figure 9 As shown, the yield of FR901379 from the above recombinant strains was lower than that of the original strain B9.

[0087] Example 5: Detection of the fermentation process of the high-yielding FR901379 recombinant strain MG5 of *Pittosporum tobira* and the control strain.

[0088] The high-yielding FR901379-producing *Pteris vittata* recombinant strain MG5 obtained by overexpressing the McfG gene in Example 1, and the recombinant strain CE-2 from patent application CN117568378A, were subjected to fermentation process testing, including data on yield, pH, and biomass. Strains MG5 and CE-2 were inoculated into 50 mL of seed culture medium and cultured at 25°C and 240 rpm for 2 days. Then, they were inoculated into 50 mL of fermentation culture medium at a volume concentration of 10% and cultured at 25°C and 240 rpm for 13 days. At fixed time points within 3-13 days, appropriate amounts of fermentation broth were collected for pH, biomass, and FR901379 yield measurement. The data are as follows: Figure 10 As shown in the figure, Figure a represents the fermentation of the control strain CE-2, and Figure b represents the fermentation of the recombinant strain MG5. While there was no significant difference in maximum yield between strain MG5 and the control strain, the fermentation cycle was significantly shortened from 10 days to 8 days. This strain achieves significant optimization of the fermentation cycle while ensuring stable fermentation capacity, resulting in better overall unit-time capacity and comprehensive production cost, demonstrating clear industrial value.

Claims

1. A highly efficient engineered fungus for producing FR901379, characterized in that, The engineered strain of *Pyrrosia lingua* is prepared by overexpressing the 3S-hydroxy-L-homocysine synthesis gene in the *Pyrrosia lingua* genome. McfG ATP-binding cassette ABC transporter synthesis gene McfM Or 3-hydroxy-L-glutamine synthesis gene McfN One of the selected strains was *Pittosporum tobira*, which showed a significant increase in yield.

2. The engineered fungus for *Pittosporum tobira* as described in claim 1, characterized in that, The McfG The nucleotide sequence is shown in SEQ ID NO. 2; McfM The nucleotide sequence is shown in SEQ ID NO.3; McfN The nucleotide sequence is shown in SEQ ID NO.

4.

3. The engineered fungus for *Pittosporum tobira* as described in claim 1, characterized in that, The McfG , McfM as well as McfN All were regulated by the gpdA promoter derived from Aspergillus nidulans.

4. The engineered fungus for *Pittosporum tobira* as described in claim 3, characterized in that, The gpdA promoter nucleotide sequence is shown in SEQ ID NO.

1.

5. The engineered fungus for *Pittosporum tobira* as described in claim 1, characterized in that, The engineered strain of *Pyrhoa sheathis* was constructed as follows: (1) The target gene and promoter were ligated to the restriction endonuclease of the vector pDHt. SpeI and XbaI (1) Construct a vector containing the target gene between the loci; (2) Transform the vector containing the target gene into Agrobacterium using Agrobacterium transformation method and prepare Agrobacterium bacterial solution; (3) Mix the Agrobacterium bacterial solution from step (2) with the suspension of Pterygotic spores, spread them on IM plates for co-culture, screen positive transformants with a significant increase in FR901379 yield, and obtain the engineered Pterygotic spore strain.

6. The engineered fungus for *Pittosporum tobira* as described in claim 5, characterized in that, Step (2) The Agrobacterium is AGL1 Agrobacterium.

7. The engineered fungus for *Pittosporum tobira* as described in claim 5, characterized in that, Step (2) The method for preparing Agrobacterium culture is as follows: Inoculate positive Agrobacterium strains into LB solid medium and culture at 28℃ for 60 h; pick colonies into LB liquid medium and culture at 28℃ and 200 rpm for 48 h with shaking; centrifuge the culture medium at 4000 rpm for 5 min; resuspend the collected bacterial pellet in IM liquid induction medium to OD. 600 The concentration was set to 0.3, and then the mixture was incubated at 28°C with shaking for 6 hours until the OD value reached 0.

3. 600 1. The Agrobacterium tumefaciens culture was obtained after expansion. The IM liquid induction medium consisted of: 5 mL / L glycerol, 1 mL / L 200 μM acetylsylgenone aqueous solution, 1.8 g / L glucose, 1.181 mL / L 30% CaCl2 aqueous solution, 400 mL / L 2.5×MM salt solution, 26.875 mL / L 10% MgSO4 aqueous solution, and 40 mL / L 40 mM 2-morpholine ethanesulfonic acid aqueous solution, with natural pH. The 2.5×MM salt solution consisted of: 1.25 g / L (NH4)2SO4, 0.375 g / L NaCl, 5.125 g / L KH2PO4, and 3.75 g / L K2HPO4, with water as the solvent.

8. The engineered fungus for stem sheath mold as described in claim 5, characterized in that, Step (3) The method for preparing the spore suspension of *Pteris sheathii* is as follows: *Pteris sheathii* is inoculated onto PDA plate medium and activated at 25°C for 10 days until mature spores are produced. After that, the colony surface is scraped with sterile physiological saline. The mixture of mycelium and physiological saline is filtered through three layers of lens paper to obtain a fresh spore suspension.

9. The engineered fungus for *Pittosporum tobira* as described in claim 5, characterized in that, Step (3) Agrobacterium tumefaciens bacterial solution OD 600 The concentration of the fungal suspension in the sheath stem was 1, and the concentration of the fungal suspension was 10. 6 The number of cells / mL was 1:

1.

10. A method for high-efficiency fermentation production of FR901379 using the engineered fungus *Pyrtomyces sheathii* as described in claim 1, characterized in that, The method is as follows: The engineered fungus *Pyrtomyces sheathinarum* is inoculated into a seed culture medium and cultured at 25℃ and 240 rpm for 2 days to obtain a seed liquid. Then, the seed liquid is inoculated into a fermentation culture medium at a volume concentration of 10%, and cultured on a shaker at 25℃ and 240 rpm for 10 days to obtain a fermentation broth containing FR901379. The seed culture medium consists of: glucose 20 g / L, corn starch 10 g / L, soybean meal 10 g / L, KH₂PO₄ 2 g / L, water as solvent, and pH adjusted to 5.5 with NaOH. The fermentation culture medium consists of: glucose 10 g / L, sorbitol 100 g / L, soybean meal 18 g / L, triammonium citrate 3 g / L, (NH₄)₂SO₄ 3 g / L, KH₂PO₄ 1 g / L, MgSO₄ 2 g / L, MnSO₄ 0.5 g / L, FeSO₄ 0.3 g / L. g / L, CaCO3 4 g / L, solvent is water, pH value is normal.

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