Strain with high yield of fr901379 and construction method and application thereof

Abstract

The application discloses a genetically engineered strain for high-yield of micafungin precursor FR901379, wherein the genetically engineered strain is a genetically engineered strain for overexpression of cytochrome P450 monooxygenase, and the starting strain of the genetically engineered strain is a phoma fungus. The genetically engineered strain obtained in the application can yield micafungin precursor FR901379 at a high yield, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a genetically engineered strain of the genus *Pyroderma* that increases the yield of FR901379 by overexpressing the target gene, and its construction method and application. Background Technology

[0002] Echinocandins are a class of cyclic lipopeptide derivatives of natural products that selectively inhibit the activity of β-1,3-glucan synthase in fungal cell walls, thereby affecting fungal cell wall synthesis and leading to fungal cell lysis and death. Echinocandins have a unique mechanism of action, high safety profile, broad antibacterial spectrum, and are effective against drug-resistant fungi. With increasing fungal resistance, the proportion of echinocandins in the global antifungal drug market has been rising year by year. Currently, clinically used echinocandins include caspofungin, micafungin, and anidulafungin. Among them, micafungin has unique characteristics; its precursor FR901379 has a sulfonyl group, resulting in excellent water solubility and thus improved bioavailability.

[0003] The industrial production of micafungin involves three steps: first, fermentation of *Coleophoma empetri* produces FR901379; then, fermentation with *Streptomyces* var. *fusca* hydrolyzes the fatty acid side chains; finally, chemical modification is used to add a methyl 4-(5-(4-(pentyloxy)phenyl)-3-isoxazolyl)benzoate side chain to ultimately produce micafungin. The fermentation of *C. empetri* to produce FR901379 is a crucial step in the production process, essential for cost control and post-processing. Currently, optimization of *C. empetri* production performance is generally achieved through fermentation medium optimization and physical mutagenesis; there are no reports of improving strain production performance through genetic modification. Besides FR901379, *C. empetri* fermentation products also include two other byproducts, WF11899B and WF11899C. The biggest difference between these byproducts and FR901379 lies in the degree of hydroxylation modification at the C-4 position of L-ornithine and L-homotyrosine. The fermentation of *C. empetri* results in low yields and abundant byproducts, making subsequent separation and purification difficult and keeping the production cost of micafungin high. Currently, there is an urgent need to solve these problems in the industrial production of micafungin. However, the encoding genes of the enzymes responsible for the hydroxylation of L-ornithine at C-4 and L-homotyrosine at C-4 are still unknown, which greatly limits related metabolic engineering modifications. Therefore, identifying the oxidases responsible for the hydroxylation of L-ornithine at C-4 and L-homotyrosine at C-4 will provide a target for metabolic engineering modifications, reduce byproduct accumulation, increase the yield of FR901379, and promote the improvement of micafungin production technology in terms of quality and efficiency. Summary of the Invention

[0004] The present invention provides a genetically engineered strain, wherein the starting strain of the genetically engineered strain is a fungus of the genus *Pyrodermus*.

[0005] The fungi in the genus *Coleophoma* include *Coleophoma* sp. or *C. empetri*.

[0006] In one specific embodiment, the fungus of the genus *Coleophoma* is *Coleophoma* sp. MEFC009, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 21058, deposited on November 18, 2020. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China. The telephone number is 010-64807355.

[0007] In this invention, the cytochrome P450 monooxygenases McfF and McfH in Coleophoma sp. MEFC009 were genetically modified to obtain different genetically engineered strains. Some of these strains can produce high yields of micafungin precursor FR901379.

[0008] In this invention, McfF is cytochrome P450 monooxygenase, its amino acid sequence is shown in SEQ ID No. 2, and its nucleic acid sequence is shown in SEQ ID No. 1. McfH is cytochrome P450 monooxygenase, its amino acid sequence is shown in SEQ ID No. 4, and its nucleic acid sequence is shown in SEQ ID No. 3.

[0009] In this invention, the cytochrome P450 monooxygenase is also referred to as P450 enzyme.

[0010] On one hand, the present invention provides a cytochrome P450 monooxygenase. In one embodiment, the cytochrome P450 monooxygenase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID No. 2 or 4; preferably, the cytochrome P450 monooxygenase is derived from a fungus of the genus *Coleophoma*; more preferably, the amino acid sequence of the cytochrome P450 monooxygenase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID No. 2 or 4, and the cytochrome P450 monooxygenase is derived from a fungus of the genus *Coleophoma*. The *Coleophoma* fungus includes *Coleophoma* sp. or *Coleophoma empetri*, for example, *Coleophoma* sp. MEFC009.

[0011] In a preferred embodiment, the amino acid sequence of the cytochrome P450 monooxygenase is shown in SEQ ID No. 2 or SEQ ID No. 4.

[0012] On the other hand, the present invention also provides biological materials comprising the above-mentioned cytochrome P450 monooxygenase or its encoding gene. The biological materials are selected from: vectors comprising the above-mentioned cytochrome P450 monooxygenase, or host cells comprising the above-mentioned cytochrome P450 monooxygenase.

[0013] On the other hand, the present invention also provides a gene encoding the above-mentioned cytochrome P450 monooxygenase.

[0014] On the other hand, the present invention also provides a vector containing the above-mentioned genes, or a host cell containing the vector.

[0015] In one embodiment, the vector includes a cloning vector and an expression vector, such as pET series vectors (e.g., pET-14, pET-21, pET-22, pET-28, pET-30, pET-42, pET-GST, pET-His, pET-Trx, pET-GST, pET-CKS, pET-DsbA), pMAL series vectors (e.g., pMAL-2c), and pGEX series vectors (e.g., pGEX-4T-2, pGEX-6T-1). pBAD series vectors (such as pBAD-His, pBAD-Myc), pMBP series vectors (pMBP-P, pMBP-C), pTYB2, pQE-9, pACYCDuet-1, pCDFDuet-1, pColADuet-1, pRSFDuet-1, pllP-OmpA, pUC series vectors (such as pUC18, pUC19), pQE-30, pXH2-1, pXH-43, pTRII, pGSF957.

[0016] In one embodiment, the host cell is selected from Escherichia coli (e.g., Escherichia coli DH5α, Escherichia coli BL21(DE3), Rosetta(DE3), Codon Plus(DE3)-RIPL, BL21 Codon plus(DE3), Top 10, JM109), yeast (e.g., Saccharomyces cerevisiae, Pichia pastoris, Yersinia esculenta), and Pyrtomyces scabra.

[0017] On the other hand, the present invention also provides the use of the above-mentioned cytochrome P450 monooxygenase, its encoding gene, the vector containing the gene, the above-mentioned host cell, or the above-mentioned biological material in the preparation of micafungin precursor FR901379.

[0018] On the other hand, the present invention also provides the application of the above-mentioned cytochrome P450 monooxygenase, its encoding gene, the vector containing the gene, the above-mentioned host cell, or the above-mentioned biological material in the preparation of a genetically engineered strain that produces high-yield micafungin precursor FR901379; preferably, the starting strain of the genetically engineered strain is a fungus of the genus *Pyrodermus*.

[0019] The fungi in the genus *Coleophoma* include *Coleophoma* sp. or *C. empetri*.

[0020] In one specific embodiment, the fungus of the genus *Coleophoma* is *Coleophoma* sp. MEFC009, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 21058, deposited on November 18, 2020. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China. The telephone number is 010-64807355.

[0021] The genetically engineered strain that produces high-yield micafungin precursor FR901379 is prepared by introducing the aforementioned cytochrome P450 monooxygenase into the starting strain; preferably, the introduction is overexpression.

[0022] The "introduction" includes the step of expressing the target gene in the starting strain, preferably overexpressing it. For example, the target gene is constructed into an expression vector, and the expression vector is transferred into a host cell to express the target gene, preferably overexpressing it. In other embodiments, the "introduction" includes inserting the target gene into the genome of the host cell; preferably, the insertion into the host cell genome can be achieved using a homologous recombination double exchange method; in one embodiment, the target gene and homologous arms can be inserted into a vector, and then the vector can be transferred into a host cell, utilizing the homologous arms to undergo homologous recombination double exchange with the host cell genome to insert the target gene into a suitable genomic position; in other embodiments, gene editing methods can also be used, for example, using the CRIspR / Cas system to cut at the desired genomic site, while simultaneously inserting the target gene as a foreign donor into the cutting site.

[0023] On the other hand, the present invention also provides the application of the above-mentioned genetically engineered strain in the production of micafungin precursor FR901379.

[0024] On the other hand, the present invention also provides a method for preparing micafungin precursor FR901379, the method comprising a fermentation step using the above-mentioned genetically engineered strain; optionally, the method further comprises a step of isolating / purifying FR901379.

[0025] In this invention, FR901379 is a precursor of micafungin, and its structural formula is shown in formula (I):

[0026]

[0027] In this invention, the structural formulas of WF11899B and WF11899C are shown in formula (II) and formula (III), respectively:

[0028]

[0029]

[0030] This invention also provides a compound with the structural formula shown in formula (IV):

[0031]

[0032] On the other hand, the present invention also provides a genetically engineered bacterium that produces a high yield of micafungin precursor FR901379. The genetically engineered bacterium is a genetically engineered bacterium that overexpresses the above-mentioned cytochrome P450 monooxygenase, and the starting strain of the genetically engineered bacterium is a fungus of the genus *Pyrodermus*.

[0033] In one embodiment, the genetically engineered bacteria overexpress a first cytochrome P450 monooxygenase; the first cytochrome P450 monooxygenase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID No. 2; preferably, the first cytochrome P450 monooxygenase is derived from a fungus of the genus *Pyroderma*; more preferably, the amino acid sequence of the first cytochrome P450 monooxygenase has at least 70% sequence identity with SEQ ID No. 2, and the first cytochrome P450 monooxygenase is derived from a fungus of the genus *Pyroderma*.

[0034] In one embodiment, the genetically engineered bacteria overexpress a second cytochrome P450 monooxygenase; the second cytochrome P450 monooxygenase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID No. 4; preferably, the second cytochrome P450 monooxygenase is derived from fungi of the genus *Pyroderma*; more preferably, the amino acid sequence of the second cytochrome P450 monooxygenase has at least 70% sequence identity with SEQ ID No. 4, and the second cytochrome P450 monooxygenase is derived from fungi of the genus *Pyroderma*.

[0035] In one embodiment, the genetically engineered bacteria simultaneously overexpress the first cytochrome P450 monooxygenase and the second cytochrome P450 monooxygenase.

[0036] In this invention, "overexpression" refers to the expression level or activity of the target gene in the genetically engineered bacteria being higher than that in the wild-type starting strain. In one embodiment, the overexpression can be achieved by introducing an expression vector to overexpress the target gene; in other embodiments, the overexpression can also be achieved by introducing additional copies of the target gene into the starting strain or by increasing the copy number of the target gene; in other embodiments, it can also be achieved by optimizing the promoter of the target gene, for example, by replacing the original promoter of the target gene with a promoter with higher promoter activity to achieve overexpression of the target gene.

[0037] On the other hand, the present invention also provides a method for preparing / producing micafungin precursor FR901379 using the above-mentioned genetically engineered strain, the method comprising the step of culturing the above-mentioned genetically engineered strain; or, the application of the above-mentioned genetically engineered strain in the preparation / production of micafungin precursor FR901379.

[0038] On the other hand, the present invention also provides a method for preparing / producing micafungin precursor FR901379, the method comprising the step of culturing using the above-mentioned genetically engineered strain; preferably, the method further comprises the step of isolating / purifying FR901379.

[0039] On the other hand, the present invention also provides a genetically engineered strain that produces high levels of WF11899B, wherein WF11899B is as shown in Formula II above, and the starting strain of the genetically engineered strain is a fungus of the genus *Pyrodermus*. Further, the genetically engineered strain that produces high levels of WF11899B is a genetically engineered strain obtained by mutating the cytochrome P450 monooxygenase in the starting strain; the cytochrome P450 monooxygenase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID No. 2.

[0040] On the other hand, the present invention also provides a method for preparing / producing WF11899B using the above-mentioned genetically engineered strain, the method comprising the step of culturing the above-mentioned genetically engineered strain; or, the application of the above-mentioned genetically engineered strain in the preparation / production of WF11899B.

[0041] On the other hand, the present invention also provides a method for preparing / producing WF11899B, the method comprising the step of culturing using the above-mentioned genetically engineered strain; preferably, the method further comprises the step of isolating / purifying WF11899B.

[0042] On the other hand, the present invention also provides a genetically engineered strain that produces the compound shown in Formula IV at high yield, wherein the starting strain of the genetically engineered strain is a fungus of the genus *Pyrodactylus*. Further, the genetically engineered strain that produces the compound shown in Formula IV at high yield is a genetically engineered strain obtained by mutating the cytochrome P450 monooxygenase in the starting strain; the cytochrome P450 monooxygenase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID No. 4.

[0043] On the other hand, the present invention also provides a method for preparing / producing the compound shown in Formula IV using the above-mentioned genetically engineered strain, the method comprising the step of culturing the above-mentioned genetically engineered strain; or, the application of the above-mentioned genetically engineered strain in the preparation / production of the compound shown in Formula IV.

[0044] On the other hand, the present invention also provides a method for preparing / producing the compound shown in Formula IV, the method comprising the step of culturing using the above-mentioned genetically engineered strain; preferably, the method further comprises the step of isolating / purifying the compound shown in Formula IV.

[0045] The mutations described in this invention include those resulting in loss of gene function or activity through gene deletion, gene insertion, or gene substitution.

[0046] In a preferred embodiment, gene mutation is performed by knocking out the target gene.

[0047] In one embodiment, the gene mutation can be achieved using conventional techniques in the art, such as gene knock-in or gene knockout through homologous recombination to cause loss of gene function or activity; or, gene editing techniques, such as zinc finger endonuclease (ZFN), transcription activator effector nuclease (TALEN), or CRIspR technology, can be used to mutate the above-mentioned gene to cause loss of gene function or activity.

[0048] On the other hand, the present invention also provides a method for constructing the above-mentioned genetically engineered bacteria. Attached Figure Description

[0049] Figure 1 The results are genomic PCR verification results of transformants obtained by knocking out the mcfF gene; among them, 2#, 3#, and 5# are transformants with mcfF gene deletion, and WT is the control strain Coleophoma sp.MEFC009.

[0050] Figure 2The results of HPLC analysis of fermentation products of the mcfF gene-deleted strain Coleophoma sp.-ΔmcfF are shown. Coleophoma sp.-ΔmcfF is a strain with the mcfF gene deletion, and WT is the control strain Coleophoma sp.MEFC009; 1: FR901379; 2: WF11899B; 3: WF11899C.

[0051] Figure 3 The chemical structures of compounds FR901379, WF11899B, and WF11899C are given.

[0052] Figure 4 The results are genomic PCR verification results of transformants obtained by knocking out the mcfH gene; among them, 6#, 8#, and 9# are transformants with the mcfH gene deleted, and WT is the control strain Coleophoma sp.MEFC009.

[0053] Figure 5 The results of HPLC analysis of fermentation products of the mcfH gene-deleted strain Coleophoma sp.-ΔmcfH are shown. Coleophoma sp.-ΔmcfH is a strain with the mcfH gene deletion, and WT is the control strain Coleophoma sp.MEFC009; 1: FR901379; 2: WF11899B; 3: WF11899C.

[0054] Figure 6 The results of LC-MS analysis of compound 4 in the fermentation product of the mcfH gene-deleted strain Coleophoma sp.-ΔmcfH.

[0055] Figure 7 The chemical structure of compound 4 in the fermentation product of the mcfH-deleted strain Coleophoma sp.-ΔmcfH;

[0056] Figure 8 Genomic PCR verification of transformants obtained by overexpressing the P450 enzyme McfF; where 1-23 are transformants, and WT is wild-type Coleophoma sp.MEFC009.

[0057] Figure 9 HPLC analysis results of fermentation products of engineered strains overexpressing P450 enzyme McfF and Coleophoma sp. MEFC009; where 1: FR901379; 2: WF11899B; 3: WF11899C.

[0058] Figure 10To analyze the yield of FR901379 (WF11899A), WF11899B and WF11899C in the fermentation broth of engineered strains overexpressing P450 enzyme McfF and Coleophoma sp. MEFC009;

[0059] Figure 11 Genomic PCR verification of transformants obtained by overexpressing the P450 enzyme McfH; where 1-23 are transformants, and WT is wild-type Coleophoma sp.MEFC009.

[0060] Figure 12 HPLC analysis results of fermentation products of engineered strains overexpressing P450 enzyme McfH and Coleophoma sp. MEFC009; where 1: FR901379; 2: WF11899B; 3: WF11899C.

[0061] Figure 13 To analyze the yield of FR901379 (WF11899A), WF11899B and WF11899C in the fermentation broth of engineered strains overexpressing P450 enzyme McfH and Coleophoma sp. MEFC009.

[0062] Figure 14 Genomic PCR verification of transformants obtained by simultaneous overexpression of P450 enzymes McfF and McfH; where 1-18 are transformants, and WT is wild-type Coleophoma sp.MEFC009.

[0063] Figure 15 To analyze the yield of FR901379 (WF11899A, WF11899B and WF11899C) in the fermentation broth of engineered strains that simultaneously overexpress P450 enzymes McfF and McfH and Coleophoma sp. MEFC009. Detailed Implementation

[0064] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the embodiments. Unless otherwise specified, the materials, reagents, instruments and methods used in the following embodiments are all conventional materials, reagents, instruments and methods in the art, and can be obtained through commercial channels.

[0065] In this invention, plasmid extraction was performed using OMEGA's Plasmid Mini Kit I reagent (D6942-01), and PCR fragment purification was performed using OMEGA's DNA fragment recovery Cycle-Pure Kit (D6492-01). One-step cloning enzyme... The Ultra One Step Cloning Kit was purchased from Nanjing Vazyme Company.

[0066] Seed culture medium: 15 g / L soluble starch, 10 g / L sucrose, 5 g / L cottonseed meal, 10 g / L peptone, 1 g / L KH2PO4, 2 g / L CaCO3.

[0067] Fermentation medium: 30 g / L corn starch, 30 g / L peptone, 6 g / L (NH4)2SO4, 1 g / L KH2PO4, 0.3 g / L FeSO4·7H2O, 0.01 g / L ZnSO4·7H2O, 2 g / L CaCO3.

[0068] STC: 1M sorbitol, 50mM Tris-HCl (pH 8.0), 50mM CaCl2.

[0069] PSTC: 40% PEG4000, 1M sorbitol, 50mM Tris-HCl (pH 8.0), 50mM CaCl2.

[0070] Top layer agar: PDB, 1M sorbitol and 4g / L agarose, sterilized and incubated at 45-48℃.

[0071] Regeneration screening medium plate PDA-SH: PDA plate, 1M sorbitol and 100mg / L hygromycin B.

[0072] Screening medium PDA-H: PDA plates and 100 mg / L hygromycin B.

[0073] Plasmid pXH2-1 is documented in Xuenian Huang, Xuefeng Lu, Jian-Jun Li. Cloning, characterization and application of a glyceraldehyde-3-phosphatedehydrogenase promoter from Aspergillus terreus, J Ind Microbiol Biotechnol (2014) 41:585–592.

[0074] In this embodiment, the starting strain used is a fungus of the genus *Coleophoma*, specifically *Coleophoma sp.* MEFC009. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 21058, deposited on November 18, 2020. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China. The telephone number is 010-64807355.

[0075] Example 1. Construction of an engineered strain of Coleophoma sp.-ΔmcfF with the mcfF gene knocked out.

[0076] Using the genome of wild-type Coleophoma sp. MEFC009 as a template, PCR amplification was performed using pfu DNA polymerase (Fermentas, Catalog No.: EP0501). Primers UmcfF-F (5'-cacctctaagatagtctatc-3') and UmcfF-R (5'-ctttacgcttgcgatcccgaaTGTATAAGATGCATCAGTGCC-3') amplified the upstream sequence U-mcfF, approximately 1.2 kb in size, and primers DmcfF-F (5'-cctgggttcgcaaagataattgCTCGAACGTTGGATATATAGC-3') and DmcfF-R (5'-ttgccaaaacaggctctgata-3') amplified the downstream sequence D-mcfF, also approximately 1.2 kb in size. Using plasmid pXH2-1 as a template, PCR amplification was performed using primers hph-F (5'-ttcgggatcgcaagcgtaaag-3') and hph-R (5'-caattatctttgcgaacccagg-3') to obtain a hygromycin resistance selection fragment hph with a size of approximately 2.2 kb. The hph fragment, upstream sequence U-mcfF, and downstream sequence D-mcfF were fused by fusion PCR. Using the fusion product as a template, nested primers UmcfF-CS-F (5'-ggatactttcaattatgcggcc-3') and DmcfF-CS-R (5'-aattgagggacagtcattct-3') were used to amplify a 4.4 kb knockout targeting element UmcfF-hph-DmcfF.

[0077] Using *Coleophoma* sp. MEFC009 as the starting strain, a small amount of mycelium was first taken from a PDA plate and homogenized using a hand homogenizer. 1 ml of seed culture was inoculated into 50 ml of seed culture medium and cultured in a 250 ml Erlenmeyer flask at 220 rpm and 25°C on a shaker. After 2 days, the mycelium was collected by centrifugation at 5000 rpm and 4°C for 5 min. The mycelium was homogenized again, and 0.5 ml-2 ml of seed culture was inoculated into 50 ml of seed culture medium. Cultured under the same conditions for 1 day, the medium and mycelium were then transferred to a 50 ml sterile centrifuge tube and centrifuged at 5000 rpm to collect the mycelium. The mycelium was washed twice with 0.6 M MgSO4. After washing until the mycelium turned white, 1 g of mycelium was weighed and added to 10 ml of enzymatic hydrolysis solution. The mixture was treated at 30°C and 100 rpm for 1-4 h. The enzymatic hydrolysate consisted of 1% cellulase, 0.6% lysozyme, 0.6% snailase, and 0.6M MgSO4, and was sterilized by filtration through a 0.22μm sterile filter. The protoplast reaction solution was then filtered through a sterile filter cloth. Protoplasts were collected by centrifugation at 5000 rpm and 4°C. The protoplasts were washed once with ice-cold STC, resuspended in pre-chilled STC, and the concentration was adjusted to 5 × 10⁻⁶ using STC. 7 Protoplast suspension was obtained by 1 / mL.

[0078] Add 10 μl of the UmcfF-hph-DmcfF fragment to 140 μl of the above protoplast suspension, then add 50 μl of PSTC, mix gently, and incubate on ice for 30 min. Add 1 ml of PSTC, mix well, and let stand at room temperature for 20 min; then mix with 10 ml of top agar and pour onto 3 PDA-SH regeneration screening medium plates, and incubate at 30°C in the dark for 5-7 days to obtain transformants.

[0079] Hygromycin-resistant transformants were picked from transformation selection plates and transferred to PDA-H plates. They were then passaged at 25°C for 5-7 days, with three consecutive passages. Three stable transformants (2#, 3#, and 5#) were selected for single-spore isolation and purification, and their genomes were extracted. PCR verification of the transformant genomes was performed using external primers UmcfF-F (5'-cacctctaagatagtctatc-3') and DmcfF-R (5'-ttgccaaaacaggctctgata-3'). Transformants amplifying a band of approximately 4.7 kb were considered positive, while *Coleophoma* sp. MEFC009 only amplified a band of approximately 3.2 kb. Figure 1The results showed that transformants #2, #3, and #5 were positive transformants, indicating that homologous recombination occurred at the mcfF gene position, integrating the exogenous fragment UmcfF-hph-DmcfF. The positive strain was defined as Coleophoma sp.-ΔmcfF.

[0080] Example 2. Fermentation verification of the engineered strain Coleophoma sp.-ΔmcfF with gene mcfF deletion.

[0081] Three positive engineered strains (2#, 3#, and 5#) and the control strain *Coleophoma* sp. MEFC009 were inoculated onto PDA solid plates and incubated at 25°C for 5-7 days. A small amount of mycelium was picked and extracted using a nucleic acid extractor. -24) Disrupt the mycelium and inoculate it into 50 ml of Coleophoma sp. seed culture medium (250 ml Erlenmeyer flask). Incubate at 25°C, 220 rpm, and shake for 45-48 h. Transfer 5 ml of the above-mentioned seed culture to 50 ml of Coleophoma sp. fermentation medium (250 ml Erlenmeyer flask). Incubate at 25°C, 220 rpm, and shake for 8 days. Set up 3 replicates for each strain. Take 1 ml of each fermentation broth, add an equal volume of methanol, and sonicate for 1 h. Centrifuge and collect the supernatant. Filter the sample through a 0.22 μm organic filter and analyze the processed sample by HPLC.

[0082] The HPLC analysis method was as follows: The liquid chromatography column was an Agilent C-18 reverse-phase column 883975-902 (4.6 × 150 mm, 5 μm); the mobile phase was A: 0.05% (v / v) trifluoroacetic acid aqueous solution, and mobile phase B: 0.05% (v / v) trifluoroacetic acid acetonitrile solution; the flow rate was 1 mL / min; the UV detection wavelength was 210 nm; the temperature was 30 °C; and the total elution time was 37 min. Gradient elution conditions were applied: from 0 to 5 min, the volume of mobile phase B linearly increased from 5% to 24%; from 5 to 35 min, the volume of mobile phase B linearly increased from 24% to 62%; and from 35 to 37 min, the volume of mobile phase B linearly increased from 62% to 100%. Results are as follows: Figure 2 As shown; compared with the starting strain, compound 1 (FR901379) in Coleophoma sp.-ΔmcfF disappeared, and the yield of the corresponding compound 2 (WF11899B) was the sum of compounds 1 and 2 in the starting strain Coleophoma sp. Furthermore, compared with FR901379, compound WF11899B lacked a hydroxyl group at the C-4 position of L-homotyrosine (e.g., Figure 3As shown in the figure, the P450 enzyme encoded by the gene mcfF is responsible for the hydroxylation of L-high tyrosine C-4. The nucleic acid sequence of this gene is shown in SEQ ID No. 1, and the amino acid sequence it encodes is shown in SEQ ID No. 2.

[0083] Example 3. Construction of an engineered strain of Coleophoma sp.-ΔmcfH with the mcfH gene knocked out.

[0084] Using the genome of wild-type Coleophoma sp. MEFC009 as a template, PCR amplification was performed using pfu DNA polymerase (Fermentas, Catalog No.: EP0501). Primers UmcfH-F (5'-gtgagtgttcctcaaggcag-3') and UmcfH-R (5'-ctttacgcttgcgatcccgaaATCACCGATCAGACCATCTC-3') amplified the upstream sequence U-mcfH of approximately 1.5 kb, while primers DmcfH-F (5'-cctgggttcgcaaagataattgCGCCAAGTTGTCAGCCCAAA-3') and DmcfH-R (5'-ccgctttaatcaacttggca-3') amplified the downstream sequence D-mcfH of 1.4 kb. Using plasmid pXH2-1 as a template, PCR amplification was performed using primers hph-F (5'-ttcgggatcgcaagcgtaaag-3') and hph-R (5'-caattatctttgcgaacccagg-3') to obtain a hygromycin resistance selection fragment hph with a size of approximately 2.2 kb. The hph fragment, upstream sequence U-mcfH, and downstream sequence D-mcfH were fused by fusion PCR. Using the fusion product as a template, nested primers UmcfH-CS-F (5'-atagcctattcatgatttct-3') and DmcfH-CS-R (5'-tacgcccgagcgacccgagt-3') were used to amplify a 4.8 kb knockout targeting element UmcfH-hph-DmcfH.

[0085] Using *Coleophoma* sp. MEFC009 as the starting strain, a small amount of mycelium was first taken from a PDA plate and homogenized using a hand homogenizer. 1 ml of seed culture was inoculated into 50 ml of seed culture medium and cultured in a 250 ml Erlenmeyer flask at 220 rpm and 25°C on a shaker. After 2 days, the mycelium was collected by centrifugation at 5000 rpm and 4°C for 5 min. The mycelium was homogenized again, and 0.5 ml-2 ml of seed culture was inoculated into 50 ml of seed culture medium. Cultured under the same conditions for 1 day, the medium and mycelium were then transferred to a 50 ml sterile centrifuge tube and centrifuged at 5000 rpm to collect the mycelium. The mycelium was washed twice with 0.6 M MgSO4. After washing until the mycelium turned white, 1 g of mycelium was weighed and added to 10 ml of enzymatic hydrolysis solution. The mixture was treated at 30°C and 100 rpm for 1-4 h. The enzymatic hydrolysate consisted of 1% cellulase, 0.6% lysozyme, 0.6% snailase, and 0.6M MgSO4, and was sterilized by filtration through a 0.22μm sterile filter. The protoplast reaction solution was then filtered through a sterile filter cloth. Protoplasts were collected by centrifugation at 5000 rpm and 4°C. The protoplasts were washed once with ice-cold STC, resuspended in pre-chilled STC, and the concentration was adjusted to 5 × 10⁻⁶ using STC. 7 Protoplast suspension was obtained by 1 / mL.

[0086] Add 10 μl of the UmcfH-hph-DmcfH fragment to 140 μl of the above protoplast suspension, then add 50 μl of PSTC, mix gently, and incubate on ice for 30 min. Add 1 ml of PSTC, mix well, and let stand at room temperature for 20 min; then mix with 10 ml of top agar and pour onto 3 PDA-SH regeneration screening medium plates, and incubate at 30°C in the dark for 5-7 days to obtain transformants.

[0087] Hygromycin-resistant transformants were picked from transformation selection plates and transferred to PDA-H plates. They were then passaged at 25°C for 5-7 days, with three consecutive passages. Three stable transformants (6#, 8#, and 9#) were selected for single-spore isolation and purification, and their genomes were extracted. PCR verification of the transformant genomes was performed using external primers UmcfH-F (5'-gtgagtgttcctcaaggcag-3') and DmcfH-R (5'-ccgctttaatcaacttggca-3'). Transformants amplifying a band of approximately 5.0 kb were considered positive, while *Coleophoma* sp. MEFC009 only amplified a band of approximately 3.7 kb. Figure 4The positive transformants in transformants #6, #8, and #9 indicate that homologous recombination occurred at the mcfH gene position, integrating the exogenous fragment UmcfH-hph-DmcfH. The positive strain is defined as Coleophoma sp.-ΔmcfH.

[0088] Example 4. Fermentation verification of the engineered strain Coleophoma sp.-ΔmcfH with gene mcfH deletion.

[0089] Three engineered bacterial strains (6#, 8#, and 9#) and the control strain *Coleophoma* sp. MEFC009 were inoculated onto PDA solid plates and cultured at 25°C for 5-7 days. A small amount of mycelium was picked and extracted using a nucleic acid extractor. -24) Disrupt the mycelium and inoculate it into 50 ml of Coleophoma sp. seed culture medium (250 ml Erlenmeyer flask). Incubate at 25°C, 220 rpm, and shake for 45-48 h. Transfer 5 ml of the above-mentioned seed culture to 50 ml of Coleophoma sp. fermentation medium (250 ml Erlenmeyer flask). Incubate at 25°C, 220 rpm, and shake for 8 days. Set up 3 replicates for each strain. Take 1 ml of each fermentation broth, add an equal volume of methanol, and sonicate for 1 h. Centrifuge and collect the supernatant. Filter the sample through a 0.22 μm organic filter and analyze the processed sample by HPLC.

[0090] The HPLC analysis method was as follows: The liquid chromatography column was an Agilent C-18 reverse-phase column 883975-902 (4.6 × 150 mm, 5 μm); the mobile phase was A: 0.05% (v / v) trifluoroacetic acid aqueous solution, and mobile phase B: 0.05% (v / v) trifluoroacetic acid acetonitrile solution; the flow rate was 1 mL / min; the UV detection wavelength was 210 nm; the temperature was 30 °C; and the total elution time was 37 min. Gradient elution conditions were applied: from 0 to 5 min, the volume of mobile phase B linearly increased from 5% to 24%; from 5 to 35 min, the volume of mobile phase B linearly increased from 24% to 62%; and from 35 to 37 min, the volume of mobile phase B linearly increased from 62% to 100%. Results are as follows: Figure 5 As shown, compared with the starting strain Coleophoma sp. MEFC009, compounds 1, 2, and 3 disappeared in Coleophoma sp.-ΔmcfH, while compound 4 appeared.

[0091] Compound 4 was separated and purified by semi-preparative liquid chromatography (HITACHI Primaide). Preparation method: Mobile phase A: 0.05% (v / v) trifluoroacetic acid aqueous solution; mobile phase B: 0.05% (v / v) trifluoroacetic acid acetonitrile solution; flow rate: 2 mL / min; UV detection wavelength: 210 nm; 30 °C; isocratic elution; mobile phase B comprised 55% of the mobile phase volume; total elution time: 20 min. Compound 4 was analyzed by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). The LC-MS analysis method was as follows: Agilent 1290 high-performance liquid chromatography, using an Agilent Zorbax Extend-C18 C18 column (2.1 × 50 mm, 1.8 μm); the total flow rate of the mobile phase was 0.6 mL / min; mobile phase A: 0.05% (v / v) formic acid aqueous solution, mobile phase B: 0.05% (v / v) formic acid acetonitrile solution, and the total elution time was 7.0 min; the elution conditions were: gradient elution: 0–1 min, the volume of mobile phase B linearly increased from 5% to 20%; 1–6 min, the volume of mobile phase B linearly increased from 20% to 60%; 6–7 min, the volume of mobile phase B linearly increased from 60% to 100%. The results are as follows: Figure 6 As shown, the mass-to-charge ratio of compound 4 [M+H] can be determined. + 1127.5542 (C 51 H 82 N8O 18 S, theoretical value: 1127.5541). Combined with NMR analysis results, the structure of compound 4 is as follows: Figure 7 As shown. Compared to FR901379, compound 4 lacks three hydroxyl groups at both the C-4 and C-5 positions of L-homotyrosine and L-ornithine. It is known that the P450 enzyme McfF is responsible for the hydroxylation of L-homotyrosine at the C-4 position, therefore the P450 enzyme McfH is responsible for the hydroxylation of L-ornithine at the C-4 and C-5 positions. Furthermore, knocking out the mcfH gene will affect the function of the mcfF gene. The nucleic acid sequence of mcfH is shown in SEQ ID No. 3, and its encoded amino acid sequence is shown in SEQ ID No. 4.

[0092] Example 5. Construction of recombinant strain McfF overexpressing P450 enzyme

[0093] 5.1 Construction of the McfF expression cassette for overexpressing the P450 enzyme

[0094] Using plasmid pXH2-1 as a template, PCR amplification was performed using primers PgpdAt-F (5'-ccctgggttcgcaaagataattggttacactctgggaggatcc-3') and PgpdAt-R (5'-gttgtgatgattgatgagttg-3'), yielding a promoter fragment PgpdAt of approximately 0.7 kb in size; Coleophoma Using the genome of sp.MEFC009 as a template, PCR amplification was performed using primers mcfF-F (5'-caactcatcaatcatcacaacATGCTTTCAGACACGACGGC-3') and mcfF-R (5'-gatttcagtaacgttaagtggCTATTCCGTCCGCCTTCTTA-3') to obtain a fragment mcfF of approximately 1.7 kb. Using plasmid pXH2-1 as a template, PCR amplification was performed using primers TtrpC-F (5'-ccacttaacgttactgaaat-3') and TtrpC-R (5'-tacctctaaacaagtgtacc-3') to obtain a terminator fragment TtrpC of approximately 0.7 kb. Fragments PgpdAt, mcfF, and TtrpC were fused by fusion PCR to obtain the expression cassette PgpdAt-mcfF-TtrpC. Using plasmid pXH2-1 as a template, PCR amplification was performed using primers hph-F (5'-ttcgggatcgcaagcgtaaag-3') and hph-R (5'-caattatctttgcgaacccagg-3') to obtain a hygromycin resistance selection fragment hph with a size of approximately 2.2 kb.

[0095] 5.2 Co-transformation to construct recombinant strains overexpressing the P450 enzyme McfF

[0096] Using *Coleophoma* sp. MEFC009 as the starting strain, a small amount of mycelium was first taken from a PDA plate and homogenized using a hand homogenizer. 1 ml of seed culture was inoculated into 50 ml of seed culture medium and cultured in a 250 ml Erlenmeyer flask at 220 rpm and 25°C on a shaker. After 2 days, the mycelium was collected by centrifugation at 5000 rpm and 4°C for 5 min. The mycelium was homogenized again, and 0.5 ml-2 ml of seed culture was inoculated into 50 ml of seed culture medium. Cultured under the same conditions for 1 day, the medium and mycelium were then transferred to a 50 ml sterile centrifuge tube and centrifuged at 5000 rpm to collect the mycelium. The mycelium was washed twice with 0.6 M MgSO4. After washing until the mycelium turned white, 1 g of mycelium was weighed and added to 10 ml of enzymatic hydrolysis solution. The mixture was treated at 30°C and 100 rpm for 1-4 h. The enzymatic hydrolysate consisted of 1% cellulase, 0.6% lysozyme, 0.6% snailase, and 0.6M MgSO4, and was sterilized by filtration through a 0.22μm sterile filter. The protoplast reaction solution was then filtered through a sterile filter cloth. Protoplasts were collected by centrifugation at 5000 rpm and 4°C. The protoplasts were washed once with ice-cold STC, resuspended in pre-chilled STC, and the concentration was adjusted to 5 × 10⁻⁶ using STC. 7 Protoplast suspension was obtained by 1 / mL.

[0097] Add the PgpdAt-mcfF-TtrpC fragment and the hph fragment to 140 μl of the above protoplast suspension, then add 50 μl of PSTC, mix gently, and incubate on ice for 30 min. Add 1 ml of PSTC, mix well, and let stand at room temperature for 20 min; then mix with 10 ml of top agar and pour onto 3 PDA-SH regeneration screening medium plates, and incubate at 30°C in the dark for 5-7 days to obtain transformants.

[0098] Transformants with hygromycin resistance were picked from the transformation selection plate and transferred to PDA-H plates. They were then passaged at 25°C for 5-7 days, with a total of 3 passages. Twenty-three transformants were selected for isolation and purification. The purified transformant genomes were verified by PCR using primers PgpdAt-F (5'-ccctgggttcgcaaagataattggttacactctgggaggatcc-3') and TtrpC-R (5'-tacctctaaacaagtgtacc-3'). A positive transformant was one that amplified a band of approximately 3.2 kb. Figure 8 As shown; among the 23 transformants, except for transformant 21#, all the other transformants were positive transformants, and the expression element PgpdAt-mcfF-TtrpC was integrated into the genome.

[0099] Example 6. Fermentation verification of recombinant strain McfF overexpressing P450 enzyme

[0100] The engineered strain overexpressing mcfF obtained in Example 5 and the control strain Coleophoma sp. MEFC009 were inoculated onto PDA solid plates and cultured at 25°C for 5-7 days. A small amount of mycelium was picked and extracted using a nucleic acid extractor (…). -24) Disrupt the mycelium and inoculate it into 50 ml of Coleophoma sp. seed culture medium (250 ml Erlenmeyer flask). Incubate at 25°C, 220 rpm, and shake for 45-48 h. Transfer 5 ml of the above-mentioned seed culture to Coleophoma sp. fermentation medium and incubate at 25°C, 220 rpm, and shake for 8 days. Set up 3 replicates for each strain. Take 1 ml of each fermentation broth, add an equal volume of methanol, and sonicate for 1 h. Centrifuge and collect the supernatant. Filter the sample through a 0.22 μm organic filter and analyze the processed sample by HPLC.

[0101] The HPLC analysis method was as follows: The liquid chromatography column was an Agilent C-18 reverse-phase column 883975-902 (4.6 × 150 mm, 5 μm); the mobile phase was A: 0.05% (v / v) trifluoroacetic acid aqueous solution, and mobile phase B: 0.05% (v / v) trifluoroacetic acid acetonitrile solution; the flow rate was 1 mL / min; the UV detection wavelength was 210 nm; the temperature was 30 °C; and the total elution time was 37 min. Gradient elution conditions were applied: from 0 to 5 min, the volume of mobile phase B linearly increased from 5% to 24%; from 5 to 35 min, the volume of mobile phase B linearly increased from 24% to 62%; and from 35 to 37 min, the volume of mobile phase B linearly increased from 62% to 100%. The HPLC analysis results are as follows: Figure 9 As shown, compared with the wild-type Coleophoma sp. MEFC009, the engineered strain Coleophoma sp.::mcfF exhibits increased production of compound FR901379 and decreased production of byproduct WF11899B. Figure 10As shown, the yield of FR901379 in the engineered strain overexpressing mcfF was higher than that in the control strain Coleophoma sp. MEFC009. The yield of FR901379 in the engineered strain overexpressing mcfF was 706 mg / L, which was 130% higher than that in the control strain Coleophoma sp. MEFC009. In addition to Coleophoma sp. MEFC009, strain Coleophoma empetri F-11899 can also produce FR901379. We also overexpressed the homolog of mcfF, BAN91490.1 (Sequence ID in NCBI), in C. empetri F-11899, and the yield of FR901379 was 100% higher than that in the control strain C. empetri F-11899.

[0102] Example 7. Construction of a recombinant strain McfH overexpressing P450 enzyme

[0103] Using the genome of Coleophoma sp. MEFC009 as a template, PCR amplification was performed using primers mcfH-F (5'-caactcatcaatcatcacaacATGGTTCCATCAATGATCTC-3') and mcfH-R (5'-gatttcagtaacgttaagtggTCACAGGGCTACTTTCGATC-3') to obtain a fragment mcfH of approximately 1.7 kb in size. Using fusion PCR, the fragments PgpdAt and TtrpC from Example 5 were fused with the fragment mcfH to obtain the expression cassette PgpdAt-mcfH-TtrpC.

[0104] Using *Coleophoma* sp. MEFC009 as the starting strain, a small amount of mycelium was first taken from a PDA plate and homogenized using a hand homogenizer. 1 ml of seed culture was inoculated into 50 ml of seed culture medium and cultured in a 250 ml Erlenmeyer flask at 220 rpm and 25°C on a shaker. After 2 days, the mycelium was collected by centrifugation at 5000 rpm and 4°C for 5 min. The mycelium was homogenized again, and 0.5 ml-2 ml of seed culture was inoculated into 50 ml of seed culture medium. Cultured under the same conditions for 1 day, the medium and mycelium were then transferred to a 50 ml sterile centrifuge tube and centrifuged at 5000 rpm to collect the mycelium. The mycelium was washed twice with 0.6 M MgSO4. After washing until the mycelium turned white, 1 g of mycelium was weighed and added to 10 ml of enzymatic hydrolysis solution. The mixture was treated at 30°C and 100 rpm for 1-4 h. The enzymatic hydrolysate consisted of 1% cellulase, 0.6% lysozyme, 0.6% snailase, and 0.6M MgSO4, and was sterilized by filtration through a 0.22μm sterile filter. The protoplast reaction solution was then filtered through a sterile filter cloth. Protoplasts were collected by centrifugation at 5000 rpm and 4°C. The protoplasts were washed once with ice-cold STC, resuspended in pre-chilled STC, and the concentration was adjusted to 5 × 10⁻⁶ using STC. 7 Protoplast suspension was obtained by 1 / mL.

[0105] Add the PgpdAt-mcfH-TtrpC and hph fragments to 140 μl of the above protoplast suspension, then add 50 μl of PSTC, mix gently, and incubate on ice for 30 min. Add 1 ml of PSTC, mix well, and let stand at room temperature for 20 min; then mix with 10 ml of top agar and pour onto three PDA-SH regeneration screening medium plates. Incubate at 30°C in the dark for 5-7 days to obtain transformants.

[0106] Transformants with hygromycin resistance were picked from the transformation selection plate and transferred to PDA-H plates. They were then passaged at 25°C for 5-7 days, with a total of 3 passages. Twenty-three transformants were selected for isolation and purification. The purified transformant genomes were verified by PCR using primers PgpdAt-F (5'-ccctgggttcgcaaagataattggttacactctgggaggatcc-3') and TtrpC-R (5'-tacctctaaacaagtgtacc-3'). A positive transformant was one that amplified a band of approximately 3.2 kb. Figure 11 As shown, all 23 transformants were positive transformants, and the expression element PgpdAt-mcfH-TtrpC was integrated into their genome.

[0107] Example 8. Fermentation verification of the McfH recombinant strain overexpressing P450 enzyme

[0108] The engineered strain overexpressing the P450 enzyme McfH from Example 7 and the control strain Coleophoma sp. MEFC009 were inoculated onto PDA solid plates and cultured at 25°C for 5-7 days. A small amount of mycelium was picked and extracted using a nucleic acid extractor (…). -24) Disrupt the mycelium and inoculate it into 50 ml of Coleophoma sp. seed culture medium (250 ml Erlenmeyer flask). Incubate at 25°C, 220 rpm, and shake for 45-48 h. Transfer 5 ml of the above-mentioned seed culture to Coleophoma sp. fermentation medium and incubate at 25°C, 220 rpm, and shake for 8 days. Set up 3 replicates for each strain. Take 1 ml of each fermentation broth, add an equal volume of methanol, and sonicate for 1 h. Centrifuge and collect the supernatant. Filter the sample through a 0.22 μm organic filter and analyze the processed sample by HPLC.

[0109] The HPLC analysis method was as follows: The liquid chromatography column was an Agilent C-18 reverse-phase column 883975-902 (4.6 × 150 mm, 5 μm); the mobile phase was A: 0.05% (v / v) trifluoroacetic acid aqueous solution, and mobile phase B: 0.05% (v / v) trifluoroacetic acid acetonitrile solution; the flow rate was 1 mL / min; the UV detection wavelength was 210 nm; the temperature was 30 °C; and the total elution time was 37 min. Gradient elution conditions were applied: from 0 to 5 min, the volume of mobile phase B linearly increased from 5% to 24%; from 5 to 35 min, the volume of mobile phase B linearly increased from 24% to 62%; and from 35 to 37 min, the volume of mobile phase B linearly increased from 62% to 100%. The HPLC analysis results are as follows: Figure 12 As shown, compared with the wild-type Coleophoma sp. MEFC009, the engineered strain Coleophoma sp.::mcfH exhibits increased production of compound FR901379 and reduced accumulation of byproduct WF11899C, which is almost no longer produced. Figure 13 As shown, the yield of FR901379 in the engineered strain overexpressing mcfH was higher than that of the control strain Coleophoma sp. MEFC009. The yield of FR901379 in the engineered strain overexpressing mcfH was 436 mg / L, which was 44% higher than that of the control strain Coleophoma sp. MEFC009. We also overexpressed the mcfH homolog BAN91489.1 (Sequence ID in NCBI) in C. empetri F-11899, and the yield of FR901379 was 32.8% higher than that of the control strain C. empetri F-11899.

[0110] Example 9. Construction of recombinant strains simultaneously overexpressing P450 enzymes McfF and McfH

[0111] Using *Coleophoma* sp. MEFC009 as the starting strain, a small amount of mycelium was first taken from a PDA plate and homogenized using a hand homogenizer. 1 ml of seed culture was inoculated into 50 ml of seed culture medium and cultured in a 250 ml Erlenmeyer flask at 220 rpm and 25°C on a shaker. After 2 days, the mycelium was collected by centrifugation at 5000 rpm and 4°C for 5 min. The mycelium was homogenized again, and 0.5 ml-2 ml of seed culture was inoculated into 50 ml of seed culture medium. Cultured under the same conditions for 1 day, the medium and mycelium were then transferred to a 50 ml sterile centrifuge tube and centrifuged at 5000 rpm to collect the mycelium. The mycelium was washed twice with 0.6 M MgSO4. After washing until the mycelium turned white, 1 g of mycelium was weighed and added to 10 ml of enzymatic hydrolysis solution. The mixture was treated at 30°C and 100 rpm for 1-4 h. The enzymatic hydrolysate consisted of 1% cellulase, 0.6% lysozyme, 0.6% snailase, and 0.6M MgSO4, and was sterilized by filtration through a 0.22μm sterile filter. The protoplast reaction solution was then filtered through a sterile filter cloth. Protoplasts were collected by centrifugation at 5000 rpm and 4°C. The protoplasts were washed once with ice-cold STC, resuspended in pre-chilled STC, and the concentration was adjusted to 5 × 10⁻⁶ using STC. 7 Protoplast suspension was obtained by 1 / mL.

[0112] The expression cassettes PgpdAt-mcfF-TtrpC, PgpdAt-mcfH-TtrpC, and hph constructed in the aforementioned examples were added to 200 μl of the protoplast suspension, followed by 50 μl of PSTC. The mixture was gently mixed and incubated on ice for 30 min. Then, 1 ml of PSTC was added, mixed, and incubated at room temperature for 20 min. The mixture was then combined with 10 ml of top agar and poured onto three PDA-SH regeneration screening medium plates. The plates were incubated at 30°C in the dark for 5-7 days to obtain transformants.

[0113] Transformants with hygromycin resistance were picked from the transformation selection plate and transferred to PDA-H plates. They were then passaged at 25°C for 5-7 days, with a total of 3 passages. Eighteen transformants were selected for isolation and purification. The purified transformant genomes were verified by PCR using primers PgpdAt-F (5'-ccctgggttcgcaaagataattggttacactctgggaggatcc-3') and mcfF-R (5'-gatttcagtaacgttaagtggCTATTCCGTCCGCCTTCTTA-3'), PgpdAt-F (5'-ccctgggttcgcaaagataattggttacactctgggaggatcc-3') and mcfH-R (5'-gatttcagtaacgttaagtggTCACAGGGCTACTTTCGATC-3'). Transformants that simultaneously amplified a band of approximately 2.5 kb were considered positive. Figure 14 As shown; of these 18 transformants, except for transformants 1#, 16#, and 17#, the others are all positive transformants, and their genomes simultaneously integrate the expression elements PgpdAt-mcfF-TtrpC and PgpdAt-mcfH-TtrpC.

[0114] Example 10. Fermentation verification of recombinant strains simultaneously overexpressing P450 enzymes McfF and McfH

[0115] The recombinant strain that simultaneously overexpressed the P450 enzymes McfF and McfH, as described in Example 9, and the control strain Coleophomasp. MEFC009 were inoculated onto PDA solid plates and cultured at 25°C for 5-7 days. A small amount of mycelium was picked and extracted using a nucleic acid extractor (…). -24) The mycelia were disrupted, and the disrupted mycelia were inoculated into 50 ml of Coleophoma sp. seed culture medium (250 ml Erlenmeyer flask) and cultured at 25℃, 220 rpm on a shaker for 45-48 h. 5 ml of the above-mentioned seed culture was transferred to 50 ml of Coleophoma sp. fermentation medium (250 ml Erlenmeyer flask) and cultured at 25℃, 220 rpm on a shaker for 8 days. Three replicates were set up for each strain. 1 ml of each fermentation broth was taken, an equal volume of methanol was added, and the mixture was ultrasonically extracted for 1 h. The supernatant was collected by centrifugation. The sample was filtered through a 0.22 μm organic filter and analyzed by HPLC. The fermentation results are as follows: Figure 15As shown, compared with the control strain, the engineered strain Coleophoma sp.::mcfF::mcfH, which simultaneously overexpresses mcfF and mcfH, exhibited increased yield of FR901379, while simultaneously reducing the accumulation of the two byproducts WF11899B and WF11899C. Similarly, we simultaneously overexpressed the homologous genes BAN91490.1 and BAN91489.1 (Sequence ID in NCBI) of mcfF and mcfH in C. empetri F-11899. Compared with the control strain C. empetri F-11899, FR901379 showed increased yield of FR901379, while simultaneously reducing the accumulation of the two byproducts WF11899B and WF11899C.

[0116] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Without departing from the spirit and scope of the present invention, those skilled in the art can make various modifications and variations on this basis. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A genetically engineered strain that produces a high yield of micafungin precursor FR901379, wherein the genetically engineered strain is a genetically engineered strain overexpressing first-cytochrome P450 monooxygenase and second-cytochrome P450 monooxygenase, and the originating strain of the genetically engineered strain is *Pteris* genus (…). Coleophoma sp . Fungi; The amino acid sequence of the first cytochrome P450 monooxygenase is shown in SEQ ID No. 2; The amino acid sequence of the second cytochrome P450 monooxygenase is shown in SEQ ID No.

4.

2. A method for preparing / producing micafungin precursor FR901379, the method comprising the step of culturing the genetically engineered strain of claim 1.

3. The method according to claim 2, characterized in that, The method also includes the step of isolating / purifying FR901379.

4. The use of the genetically engineered strain according to claim 1 in the preparation / production of micafungin precursor FR901379.

5. A method for preparing the genetically engineered strain of claim 1, the method comprising the step of overexpressing the first cytochrome P450 monooxygenase and the second cytochrome P450 monooxygenase in the starting strain; The amino acid sequence of the first cytochrome P450 monooxygenase is shown in SEQ ID No. 2; The amino acid sequence of the second cytochrome P450 monooxygenase is shown in SEQ ID No.

4.

6. The application of the coding genes for first cytochrome P450 monooxygenase and second cytochrome P450 monooxygenase, or the coding genes for first cytochrome P450 monooxygenase and second cytochrome P450 monooxygenase, in the preparation of a genetically engineered strain producing high-yield micafungin precursor FR901379, wherein the starting strain of the genetically engineered strain is *Pteris* genus (…). Coleophoma sp . Fungi; The amino acid sequence of the first cytochrome P450 monooxygenase is shown in SEQ ID No. 2; The amino acid sequence of the second cytochrome P450 monooxygenase is shown in SEQ ID No.

4.

7. The use of the first cytochrome P450 monooxygenase and the second cytochrome P450 monooxygenase as described in claim 1 in the preparation of micafungin precursor FR901379.

8. Application of vectors containing the encoding genes of first cytochrome P450 monooxygenase and second cytochrome P450 monooxygenase in the preparation of micafungin precursor FR901379; The amino acid sequence of the first cytochrome P450 monooxygenase is shown in SEQ ID No. 2; The amino acid sequence of the second cytochrome P450 monooxygenase is shown in SEQ ID No. 4.

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