WO2024017105A1 - 提高棘白菌素类化合物产量的转录因子及其应用 - Google Patents

提高棘白菌素类化合物产量的转录因子及其应用 Download PDF

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WO2024017105A1
WO2024017105A1 PCT/CN2023/106918 CN2023106918W WO2024017105A1 WO 2024017105 A1 WO2024017105 A1 WO 2024017105A1 CN 2023106918 W CN2023106918 W CN 2023106918W WO 2024017105 A1 WO2024017105 A1 WO 2024017105A1
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strain
transcription factor
seq
genetically engineered
echinocandins
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French (fr)
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吕雪峰
黄雪年
门萍
张璇
刘永娟
周宇
谢丽
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中国科学院青岛生物能源与过程研究所
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/38—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Aspergillus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus

Definitions

  • the invention belongs to the technical field of genetic engineering and relates to a transcription factor that improves the production of echinocandins and its application. Specifically, it relates to the specific transcription regulatory factors FR901379, echinocandin B and neomer in the overexpression synthesis pathway. Genetically engineered bacteria with improved Kangding B0 production and their construction methods and applications.
  • Echinocandins prodrugs are a type of cyclic lipopeptide compounds produced by filamentous fungi. These drugs can selectively inhibit the activity of ⁇ -1,3 glucan synthase in the fungal cell wall, thereby affecting the fungi. Cell wall synthesis leads to fungal cell lysis and death. Since mammals do not have cell walls, this type of drug has little side effects on the human body and is highly safe. In addition, this type of drug is effective against drug-resistant bacteria.
  • the echinocandin antifungal drugs currently used clinically include caspofungin, micafungin and anidulafungin. Their precursor compounds are niumocandin B 0 , FR901379 and echinocandin B respectively. Their cyclic lipopeptide skeletons are synthesized by non-ribosomal peptide synthases and fatty acids or polyketide synthases.
  • the metabolic regulatory system composed of transcription factors has the ability to globally and dynamically regulate target metabolic pathways and has been widely used in metabolic engineering.
  • biosynthetic pathways of echinocandin B and nimocandin B 0 have been analyzed, no pathway-specific transcriptional regulators have been found in their biosynthetic gene clusters. Therefore, there is no information on the improvement of echinocandin B based on specific transcriptional regulators. Reports on the potency of bacteriocin compounds.
  • the present invention provides a transcription factor that improves the production of echinocandins, and the amino acid sequence of the transcription factor has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7% , 99.8% or 99.9% sequence identity.
  • the transcription factor is the transcription factor McfJ
  • the amino acid sequence of McfJ has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity
  • the McfJ source For fungi of the genus Coleophoma, for example, Coleophoma sp. MEFC009.
  • the amino acid sequence of McfJ is shown in SEQ ID No. 2.
  • the transcription factor is the transcription factor EcdJ
  • the amino acid sequence of EcdJ has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity
  • the EcdJ source For Aspergillus nidulans for example, Aspergillus nidulans ARTP-7 with deposit number CGMCC No. 40073.
  • the amino acid sequence of EcdJ is shown in SEQ ID No. 4; the above strain (Aspergillus nidulans) ARTP-7 is recorded in Chinese patent application CN114907989A.
  • the transcription factor is the transcription factor ctg12_1653, and the amino acid sequence of ctg12_1653 has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity, preferably, the source of ctg12_1653 For Glarea sp., for example, Glarea lozoyensis, for example, Glarea lozoyensis ATCC 74030.
  • the amino acid sequence of ctg12_1653 is shown in SEQ ID No. 6.
  • the present invention also provides biological materials comprising the above-mentioned transcription factors or genes encoding them.
  • the biological material is selected from: a vector containing the above-mentioned transcription factor, or a host cell containing the above-mentioned transcription factor.
  • the present invention also provides genes encoding the above-mentioned transcription factors.
  • the present invention also provides a vector containing the above gene, or a host cell containing the vector.
  • the vector includes cloning vectors and expression vectors, for example, pET series vectors (eg, 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 (such as pMAL-2c), pGEX series vectors (such as 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,
  • the host cell is selected from the group consisting of E. coli DH5 ⁇ , E. coli BL21(DE3), Rosetta(DE3), Codon Plus(DE3)-RIPL, BL21Codon plus(DE3), Top 10, JM109), yeast (e.g., Saccharomyces cerevisiae, Pichia pastoris, Yarrowia esterolytica), Phytophthora fungi, Aspergillus nidulans, Aspergillus pachycristatus, A.delacroxii, E.rugulosa, E.nidulans, or Glarea sp.
  • yeast e.g., Saccharomyces cerevisiae, Pichia pastoris, Yarrowia esterolytica
  • Phytophthora fungi Aspergillus nidulans, Aspergillus pachycristatus, A.delacroxii, E.rugulosa, E.nidul
  • the present invention also provides the use of the above-mentioned transcription factors, their encoding genes, vectors containing the genes, the above-mentioned host cells, or the above-mentioned biological materials in the preparation of echinocandin compounds.
  • the echinocandin compound is FR901379, echinocandin B or niumocandin B 0 .
  • the present invention provides the use of the transcription factor McfJ, its encoding gene, a vector containing the gene, the above-mentioned host cell, or the above-mentioned biological material in the preparation of echinocandins; the echinocandins
  • the compound is FR901379.
  • the present invention provides the use of the transcription factor EcdJ, its encoding gene, a vector containing the gene, the above-mentioned host cell, or the above-mentioned biological material in the preparation of echinocandins; the echinocandins
  • the compound is echinocandin B.
  • the present invention provides the application of the transcription factor ctg12_1653, its encoding gene, a vector containing the gene, the above-mentioned host cell, or the above-mentioned biological material in the preparation of echinocandins; the echinocandins
  • the compound is niumocontin B 0 .
  • the present invention also provides the application of the above-mentioned transcription factor, its encoding gene, a vector containing the gene, the above-mentioned host cell, or the above-mentioned biological material in preparing a genetically engineered strain with high yield of echinocandin compounds; in one implementation
  • the transcription factor is McfJ
  • the echinocandins are FR901379
  • the starting strain of the genetic engineering strain is a fungus of the genus Coleophoma, preferably Coleophoma sp.
  • the transcription factor is EcdJ
  • the echinocandin compound is echinocandin B
  • the starting strain of the genetically engineered strain is Aspergillus nidulans, Aspergillus pachycristatu, Emericella nidulans, A.delacroxii , E.rugulosa or E.nidulans
  • the starting strain is capable of producing echinocandin B, preferably Aspergillus nidulans
  • the transcription factor is ctg12_1653
  • the echinocandin compound is neon.
  • Mocandine B 0 the starting strain of the genetically engineered strain is Glarea sp., preferably, Glarea lozoyensis.
  • the above-mentioned genetically engineered strain for preparing a high-yield echinocandin compound is to introduce the above-mentioned transcription factor or its encoding gene into the starting strain; preferably, the introduction is overexpression.
  • the present invention also provides a genetically engineered strain that is highly productive of echinocandin compounds.
  • the genetically engineered strain is a genetically engineered strain obtained by overexpressing the above-mentioned transcription factor in the starting strain.
  • the transcription factor is McfJ
  • the echinocandins are FR901379
  • the starting strain of the genetically engineered strain is a fungus of the genus Coleophoma, preferably Coleophoma sp.
  • the transcription factor is EcdJ
  • the echinocandin compound is echinocandin B
  • the starting strain of the genetically engineered strain is Aspergillus nidulans, Aspergillus pachycristatu, Emericella nidulans, A.delacroxii, E.rugulosa or E.nidulans, preferably Aspergillus nidulans
  • the transcription factor is ctg12_1653
  • the echinocandin compound is niumocandin B 0
  • the genetic engineering The starting strain of the strain is Glarea sp., preferably, Glarea lozoyensis.
  • the present invention also provides a method for preparing a genetically engineered strain with high production of echinocandin compounds, which method includes the step of overexpressing the above-mentioned transcription factor in a starting strain to prepare the genetically engineered strain.
  • the transcription factor is McfJ
  • the echinocandins are FR901379
  • the starting strain of the genetically engineered strain is a fungus of the genus Coleophoma, preferably Coleophoma sp.
  • the transcription factor is EcdJ
  • the echinocandin compound is echinocandin B
  • the starting strain of the genetically engineered strain is Aspergillus nidulans, Aspergillus pachycristatu, Emericella nidulans, A.delacroxii, E.rugulosa or E.nidulans, preferably Aspergillus pachycristatu
  • the transcription factor is ctg12_1653
  • the echinocandin compound is niumocandin B 0
  • the genetic engineering The starting strain of the strain is Glarea sp., preferably, Glarea lozoyensis.
  • “Overexpression” in the present invention means that the expression amount, activity or expression level of the target gene in the genetically engineered bacteria is higher than that of the wild-type starting strain.
  • the above-mentioned overexpression can be achieved by introducing an expression vector to overexpress the target gene; in other embodiments, the above-mentioned overexpression can also be achieved by introducing additional copies of the target gene into the starting strain, by increasing the target gene The copy number can be achieved; 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 activity to achieve overexpression of the target gene. .
  • Overexpression in the present invention includes increasing the expression level of the gene by introducing additional copies of the target gene, increasing the copy number of the target gene in the cell, or increasing the expression level of the target gene by replacing the promoter of the gene.
  • the overexpression includes the step of replacing the original promoter of the transcription factor with a strong promoter; preferably, the strong promoter is the promoter PgpdAt.
  • the overexpression includes the step of introducing additional copies of the transcription factor into the starting strain.
  • the "introduction" includes constructing the gene of interest into an exogenous expression vector, and transferring the exogenous expression vector into The starting strain is used to overexpress transcription factors; preferably, the exogenous expression vector includes pXH2-1, pXH43, pTRII, and pGSF957.
  • the "introduction" includes inserting the target gene into the genome of the starting strain; preferably, the insertion into the genome can use the method of homologous recombination double exchange; in one embodiment, You can insert the target gene and homology arm into the vector, then transfer the vector into the starting strain, and use the homology arm to perform homologous recombination double exchange with the genome to insert the target gene into the appropriate genome position; in other cases,
  • gene editing can also be used, for example, the CRISPR/Cas system is used to cut at the desired genomic site, and the target gene is inserted into the cut site as an exogenous donor.
  • the additional copy of the transcription factor when an additional copy of the transcription factor is inserted into the genome of the starting strain, the additional copy of the transcription factor is expressed under the action of a strong promoter; preferably, the strong promoter is the promoter PgpdAt .
  • the strong promoter is preferably promoter PgpdAt, PcitA, PgpdA, PtrpC, Pgpk, Pmpgd.
  • the above-mentioned promoters are all promoters known in the art, such as the journal literature Huang 592.PgpdAt is disclosed. And the journal document Dave K et al. Utility of Aspergillus niger citrate synthase promoter or heterologous expression. J Biotechnol 2011,155:173–177 disclosed PcitA.
  • the present invention also provides the use of the above-mentioned genetically engineered strain with high production of echinocandin compounds in the production of echinocandin compounds.
  • the genetically engineered strain with high production of echinocandins is a genetically engineered strain obtained by overexpressing the above-mentioned transcription factor in the starting strain; in one embodiment, the transcription factor is McfJ, and the echinocandins
  • the compound is FR901379
  • the starting strain of the genetically engineered strain is a fungus of the genus Coleophoma sp., preferably Coleophoma sp.
  • the transcription factor is EcdJ, and the echinacea
  • the cannabinoid compound is echinocandin B
  • the starting strain of the genetically engineered strain is Aspergillus nidulans, Aspergillus pachycristatu, Emericella nidulans, A.delacroxii, E.rugulosa or E.nidulans, preferably Aspergillus nidulans; in others
  • the transcription factor is ctg12_1653
  • the echinocandin compound is neomocontin B 0
  • the starting strain of the genetically engineered strain is Glarea sp., preferably, Glarea lozoyensis.
  • the present invention also provides a method for preparing echinocandins, which method includes the step of fermenting the above genetically engineered strains; optionally, the method also includes isolating/purifying the echinocandins. Procedure for cannabinoids.
  • the echinocandins are FR901379
  • the genetically engineered strain is a genetically engineered strain obtained by overexpressing the above-mentioned transcription factor in the starting strain, the transcription factor is McfJ
  • the genetically engineered strain The starting strain of the strain is a fungus of the genus Coleophoma, preferably Coleophoma sp. MEFC009.
  • the echinocandin compound is echinocandin B
  • the genetically engineered strain is a genetically engineered strain obtained by overexpressing the above-mentioned transcription factor in the starting strain, and the transcription factor is EcdJ
  • the starting strain of the genetically engineered strain is Aspergillus nidulans, Aspergillus pachycristatu, Emericella nidulans, A.delacroxii, E.rugulosa or E.nidulans, preferably Aspergillus nidulans.
  • the echinocandins are neomocontin B 0
  • the genetically engineered strain is a genetically engineered strain obtained by overexpressing the above-mentioned transcription factor in the starting strain, and the transcription factor is ctg12_1653
  • the starting strain of the genetically engineered strain is Glarea sp., preferably, Glarea lozoyensis.
  • the present invention also provides a method for improving the production of echinocandin compounds in a target strain, which method includes the step of overexpressing the above-mentioned transcription factors in the target strain.
  • the echinocandins are FR901379
  • the transcription factor is McfJ
  • the target strain is a fungus of the genus Coleophoma, preferably Coleophoma sp. MEFC009.
  • the echinocandin compound is echinocandin B
  • the transcription factor is EcdJ
  • the target strain is Aspergillus nidulans, Aspergillus pachycristatu, Emericella nidulans, A.delacroxii, E.rugulosa or E. nidulans, preferably Aspergillus nidulans.
  • the echinocandins are niumocandin B 0
  • the transcription factor is ctg12_1653
  • the target strain is Glarea sp., preferably, Glarea lozoyensi.
  • Figure 1 shows the genomic PCR verification results of the transformants obtained by knocking out the mcfJ gene; among them, 2#, 5# and 8#: transformants with deleted gene mcfJ, WT: control strain Coleophoma sp.MEFC009, M: 1kb Marker.
  • FIG. 2 shows the HPLC analysis results of the fermentation product of the gene mcfJ deletion strain
  • MEFC009 MEFC-- ⁇ mcfJ is the mcfJ deletion strain
  • the control strain Coleophoma sp. MEFC009
  • 1 FR901379.
  • FIG. 3 shows the transcript level analysis of FR901379 biosynthetic genes
  • MEFC- ⁇ mcfJ is the gene mcfJ deletion strain
  • MEFC009 is the control strain Coleophoma sp.MEFC009
  • 8763_g actin encoding gene
  • other mcf genes are genes responsible for the synthesis of FR901379.
  • Figure 4 shows the genome PCR verification of the transformants obtained by replacing the promoter of the transcription regulator mcfJ; 1-8 are the transformants, and WT-1 is Coleophoma sp.MEFC- ⁇ ku80, M: 1kb Marker.
  • Figure 5 shows the transcript level analysis of the gene mcfJ in the engineered strains MEFC::mcfJ and Coleophoma sp.MEFC009 that increase the copy number of the transcriptional regulator mcfJ.
  • Figure 6 shows the HPLC analysis results of the fermentation products of the engineering strains MEFC-PgpdAt-mcfJ and MEFC::mcfJ, which replaced the promoter of the transcription regulator McfJ and increased the copy number, and the control strain Coleophoma sp.MEFC009; 1 is FR901379.
  • Figure 7 shows the production analysis of FR901379 in the fermentation broth of engineering strains MEFC-PgpdAt-mcfJ, MEFC::mcfJ and Coleophoma sp.MEFC009 overexpressing the transcriptional regulator McfJ.
  • Figure 8 Genomic PCR verification results of transformants obtained by knocking out the ecdJ gene; among them, 8# and 16# are transformants with deleted gene ecdJ, C 2 is the wild-type control, and C 3 is the positive control.
  • FIG. 9 HPLC analysis results of the fermentation product of A.nidulans- ⁇ ecdJ gene ecdJ deletion strain; ARTP-7- ⁇ ecdJ is the gene ecdJ deletion strain, and ARTP-7 is the control strain.
  • FIG. 10 Genome PCR verification of transformants obtained by overexpressing the transcriptional regulatory factor EcdJ; No. 1-4 are transformants, C 1 is a negative control, and C 2 is a positive control.
  • FIG 11 Analysis of echinocandin B production in the engineering strain overexpressing the transcriptional regulator EcdJ and the fermentation broth of A.nidulans.ARTP-7; ARTP-7 is the wild-type control, and ARTP-7::ecdJ is the overexpression of ecdJ. strains.
  • PCR fragment purification adopts OMEGA company's DNA fragment recovery Cycle-Pure Kit (D6492-01); PCR high-fidelity enzyme and one-step cloning enzyme Ultra One Step Cloning Kit was purchased from Nanjing Vazyme Company; restriction enzymes were purchased from Thermo Fisher Scientific; RNA extraction kit and RNA reverse transcription kit were purchased from TaKaRa.
  • the seed culture medium of MEFC009 15g/L soluble starch, 10g/L sucrose, 5g/L cottonseed cake powder, 10g/L peptone, 1g/L KH 2 PO 4 , 2g/L CaCO 3 .
  • Fermentation medium of MEFC009 30g/L corn starch, 30g/L peptone, 6g/L (NH 4 ) 2 SO 4 , 1g/L KH 2 PO 4 , 0.3g/L FeSO 4 ⁇ 7H 2 O, 0.01g/ L ZnSO 4 ⁇ 7H 2 O, 2g/L CaCO 3 .
  • Aspergillus nidulans ARTP-7 seed culture medium 5 ⁇ 30g/L cottonseed cake powder, 5 ⁇ 20g/L glucose, 5 ⁇ 10g/L glycerol, adjust to pH 5.0 ⁇ 7.0 with NaOH.
  • Aspergillus nidulans ARTP-7 fermentation medium 60 ⁇ 150g/L mannitol, 15 ⁇ 50g/L peanut oil, 5 ⁇ 20g/L glycerin, 5 ⁇ 20g/L soybean cake powder, 5 ⁇ 20g/L L peptone, 0.01 ⁇ 0.07g/L Fe S O 4 ⁇ 7H 2 O, 5 ⁇ 10g/L K 2 HPO 4 , 0.2 ⁇ 1g/L MgSO 4 ⁇ 7H 2 O, 0.1 ⁇ 0.8g/L MnSO 4 ⁇ H2O, 0.3 ⁇ 0.8g/L CuSO 4 ⁇ 5H 2 O, 0.1 ⁇ 0.5g/L CaCl 2 .
  • KB01 seed culture medium 20-50g glucose monohydrate, 10-30g soybean cake powder, 5-20g cottonseed cake powder, 5-20g corn steep liquor dry powder, KH 2 PO 4 0.05-0.2g, trace elements (1mL/100mL ).
  • Race element group FeSO 4 ⁇ 7H 2 O 1g, MnSO 4 ⁇ H 2 O 1g, ZnSO 4 ⁇ 7H 2 O 0.2g, CaCl 2 ⁇ 2H 2 O 0.1g, H 3 BO 3 0.056g, CuCl 2 ⁇ 2H 2 O 0.025g, (NH 4 ) 6 Mo 7 O 24 ⁇ 4H 2 O 0.01g), pH 5.3 ⁇ 5.5.
  • KB01 fermentation medium 100-150g sorbitol, 1-10g glucose monohydrate, 5-20g cottonseed meal, 20-50g gluten powder, 1-5g K 2 HPO 4 ⁇ 3H 2 O, (NH 4 ) 2 SO 4 0.2 ⁇ 2g, NaNO 3 0.2 ⁇ 2g, FeSO 4 ⁇ 7H 2 O 0.1 ⁇ 1g, L-proline 5 ⁇ 30g, L-threonine 1 ⁇ 5g, pH 6.8.
  • PSTC 40% PEG4000, 1M sorbitol, 50mM Tris-HCl (pH 8.0), 50mM CaCl2 .
  • Top agar PDB, 1M sorbitol and 4g/L agarose, incubated at 45-48°C after sterilization.
  • Regeneration screening medium plate PDA-SH PDA plate, 1M sorbitol and 100mg/L hygromycin B.
  • Screening medium PDA-H PDA plate and 100 mg/L hygromycin B.
  • Plasmid pXH2-1 is documented in Xuenian Huang, Xuefeng Lu, Jian-Jun Li. Cloning, characterization and application of a glyceraldehyde-3-phosphate dehydrogenase promoter from Aspergillus terreus, J Ind Microbiol Biotechnol (2014) 41:585–592.
  • Plasmid PU19-ZX was constructed independently by our laboratory.
  • the starting strain used is a fungus of the genus Coleophoma sp. MEFC009.
  • the above-mentioned strain is deposited in the General Microorganism Center (CGMCC) of the China Microbiological Culture Collection Committee, and the deposit number is CGMCC No. 21058, deposited on November 18, 2020, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Tel: 010-64807355.
  • Glarea lozoyensis ATCC 74030 were purchased from the American Standard Biological Collection (ATCC). In this article, Glarea lozoyensis ATCC 74030 is also called KB01.
  • Aspergillus nidulans ARTP-7 is obtained through ARTP mutagenesis using (Aspergillus nidulans) AN01 as the starting strain.
  • the strain preservation number is CGMCC No. 40073.
  • the strain was deposited on January 29, 2022.
  • General Microbiology Center of China Microbial Culture Collection Committee; the above strain (Aspergillus nidulans) ARTP-7 is recorded in Chinese patent application CN114907989A.
  • Example 1 Construction of engineering strain MEFC- ⁇ mcfJ with knockout gene mcfJ
  • mcfJ whose amino acid sequence is annotated as a protein of unknown function.
  • Transcriptome data analysis found that the transcription level of mcfJ is equivalent to that of other genes responsible for the synthesis of FR901379. It is speculated that mcfJ may be related to the synthesis of FR901379. In order to verify its function, the gene mcfJ was knocked out.
  • the nucleic acid sequence and amino acid sequence of McfJ are shown in SEQ ID No. 1 and SEQ ID No. 2 respectively.
  • pfu DNA polymerase (Fermentas, Catalog No.: EP0501) was used for PCR amplification, using primers UmcfJ-F (5'-ccggtggcttgaaagatttc-3') and UmcfJ-R ( 5'-ctttacgcttgcgatcccgaaGTTAACTACGGACATACCT-3') can amplify the upstream sequence U-mcfJ of mcfJ with a size of about 1.3kb, using primers DmcfJ-F (5'-cctgggttcgcaaagataattgCGTCAACATCGGTGATACCC-3') and DmcfJ-R (5'-aggaggactcgaaatcaaag- 3') The downstream sequence D-mcfJ of mcfJ with a size of
  • PCR amplification was performed using primers hph-F (5'-ttcgggatcgcaagcgtaag-3') and hph-R (5'-caattatctttgcgaacccagg-3') to obtain hygromycin of approximately 2.2 kb.
  • Resistance screening fragment hph fuse the hph fragment, the upstream sequence U-mcfJ and the downstream sequence D-mcfJ through fusion PCR, and then use the fusion product as a template and use the nested primer UmcfJ-CS-F (5'-gcctagcctggccatatgca-3 ') and DmcfJ-CS-R (5'-ctttatccggacctacagc-3') were amplified by PCR to amplify the knockout targeting element UmcfJ-hph-DmcfJ with a size of 4.6 kb.
  • the components of the enzymatic solution are: 1% cellulase, 0.6% wall-lysing enzyme, 0.6% helicase and 0.6M MgSO 4 , filtered and sterilized through a 0.22 ⁇ m sterile filter. Filter the above protoplast reaction solution through sterile magic filter cloth.
  • Protoplasts were collected by centrifugation at 5000 rpm and 4°C. Wash once with ice-cold STC, re-suspend the protoplasts in the pre-cooled STC, and use STC to adjust the protoplast concentration to 5 ⁇ 10 7 /mL to obtain a protoplast suspension.
  • the band that can amplify a band of approximately 4.9kb is Positive transformants, while Coleophoma sp.MEFC009 can only amplify a band with a size of about 3.4kb.
  • the results in Figure 1 show that transformants 2#, 5#, and 8# are positive transformants, indicating that something happened at the gene mcfJ position. Homologous recombination integrated the exogenous fragment UmcfJ-hph-DmcfJ, and the positive strain was defined as MEFC- ⁇ mcfJ.
  • fermentation medium 250 mL Erlenmeyer flask
  • cultured seed liquid 250 mL Erlenmeyer flask
  • shaking table 25°C, 220 rpm for 8 days.
  • Each strain is set up in 3 parallels. Take 1 mL from each bottle of fermentation broth, add an equal volume of methanol, conduct ultrasonic extraction for 1 hour, centrifuge, and take the supernatant. The samples were filtered through a 0.22 ⁇ m organic filter and analyzed by HPLC.
  • the HPLC analysis method is: the liquid chromatography column is Agilent C-18 reverse column 883975-902 (4.6 ⁇ 150mm, 5 ⁇ m); the mobile phase is A: 0.05% (volume ratio) trifluoroacetic acid aqueous solution, mobile phase B: 0.05% (Volume ratio) trifluoroacetic acid acetonitrile solution, flow rate is 1mL/min, UV detection wavelength: 210nm, 30°C, total elution time is 37min.
  • mcfJ should not be a gene involved in the formation of the FR901379 structure, it is speculated that mcfJ may be a transporter or a transcriptional regulator. However, since FR901379 is basically located within the cell, it is not involved in transport, so it is speculated that mcfJ is likely to be a transcriptional regulator.
  • RNA spin column Place the RNA spin column on a 1.5 mL RNase-free collection tube, add 50 ⁇ L of 0.1% DEPC-treated water to the center of the RNA spin column membrane, and let stand at room temperature for 5 minutes. 13) Centrifuge at 12,000 rpm for 2 minutes to elute RNA.
  • the reaction system is as follows: 2.0 ⁇ L of 5 ⁇ gDNA Eraser buffer, 1.0 ⁇ L of gDNA Eraser, 1.0 ⁇ g of total RNA. Use RNase-free water to make up the reaction system to 10 ⁇ L, 42°C, place for 5 minutes.
  • Reverse transcription Add 4.0 ⁇ L of 5 ⁇ PrimerScript II buffer, 0.5 ⁇ L of RNase inhibitor, 1.0 ⁇ L of reverse transcriptase, 1 ⁇ L of RT Primer Mix to the reaction solution in (1), and use RNase-free Make up the reaction system to 20 ⁇ L with water, place at 37°C for 15 minutes, and then at 85°C for 5 seconds.
  • the cDNA of wild-type Coleophoma sp. MEFC009 and mcfJ deletion strain were used as templates to amplify the target fragment by PCR, and the actin encoding gene 8763_g was used as a control. Fragments were analyzed by DNA gel electrophoresis. The results are shown in Figure 3. It can be seen from the PCR electrophoresis results that when mcfJ is deleted, the genes responsible for the synthesis of FR901379 are no longer transcribed, but the actin-encoding gene 8763_g can be transcribed normally. This shows that the gene mcfJ is a specific transcriptional regulator in the FR901379 synthetic gene cluster. When mcfJ is deleted, it will affect the expression of other genes.
  • PCR amplification was performed using primers UMcfJ-2F (5'-tacaagcactaatgtaatcg-3') and UMcfJ-2R (5'-tttacgcttgcgatcccgaatagcaggaatccttatataa-3'), and the size was about 1.2kb.
  • Fragment UMcfJ-2 was PCR amplified using primers DMcfJ-2F (5'-caactcatcaatcatcacaacATGCCTATGCCTATGTCTAC-3') and DMcfJ-2R (5'-ACATGCCGTGCTGGCGACAT-3') to obtain fragment DMcfJ-2 with a size of approximately 1.2kb.
  • DMcfJ-2F 5'-caactcatcaatcatcacaacATGCCTATGCCTATGTCTAC-3'
  • DMcfJ-2R 5'-ACATGCCGTGCTGGCGACAT-3'
  • the resistance screening fragment hph was PCR amplified using plasmid pXH2-1 as a template using primers PgpdAt-F (5'-gttacactctgggaggatcc-3') and PgpdAt-R (5'-gttgtgatgattgatgagttg-3').
  • the obtained size was approximately 0.75kb strong promoter element PgpdAt.
  • fusion PCR By fusion PCR, the fragments UMcfJ-2, hph, PgpdAt and DMcfJ-2 were fused, using the fusion product as a template and nested primers UMcfJ-2CS-F (5'-aatttccctgtaatacacatt-3') and DMcfJ-2CS -R(5'-ACATGCCGTGCTGGCGACAT-3') was PCR amplified to obtain the targeting element UMcfJ-2-hph-PgpdAt-DMcfJ-2 with a size of approximately 5.1kb.
  • the components of the enzymatic solution are: 1% cellulase, 0.6% wall-lysing enzyme, 0.6% helicase and 0.6M MgSO 4 , filtered and sterilized through a 0.22 ⁇ m sterile filter. Filter the above protoplast reaction solution through sterile magic filter cloth.
  • Protoplasts were collected by centrifugation at 5000 rpm and 4°C. Wash once with ice-cold STC, re-suspend the protoplasts in the pre-cooled STC, and use STC to adjust the protoplast concentration to 5 ⁇ 10 7 /mL to obtain a protoplast suspension.
  • the band with a size of about 5.3kb is a positive transformant, while the control strain can only amplify a band with a size of 2.4kb, as shown in Figure 4; except for the 1# transformant, the gene mcfJ of other transformants
  • the promoters were all replaced by PgpdAt, and these transformants were named MEFC-PgpdAt-mcfJ.
  • PCR amplification was performed using primers PMcfJ-F (5'-taatacatcatttcattcat-3') and TMcfJ-R (5'-cggcctagaaggtcgatcgc-3'), and the size of approximately 3.5kb was obtained. Fragment P-McfJ-T. In this embodiment, the promoter of McfJ is not replaced, but the copy number of McfJ is increased.
  • the components of the enzymatic solution are: 1% cellulase, 0.6% wall-lysing enzyme, 0.6% helicase and 0.6M MgSO 4 , and are filtered and sterilized through a 0.22 ⁇ m sterile filter. Filter the above protoplast reaction solution through sterile magic filter cloth.
  • Protoplasts were collected by centrifugation at 5000 rpm and 4°C. Wash once with ice-cold STC, resuspend the protoplasts in the pre-cooled STC, and use STC to adjust the protoplast concentration to 5 ⁇ 10 7 /mL to obtain a protoplast suspension.
  • MEFC009 and the mutant strain MEFC::mcfJ overexpressing McfJ were used as templates respectively, and the primers McfJCDS-F (5'-atgacgatagtatagagatggtc-3') and McfJCDS-R (5'-ggagctacacagtgaccttc-3 '), amplified by PCR, and the actin-encoding gene 8503_g was used as a control. The results are shown in Figure 5.
  • the engineering strains MEFC-PgpdAt-mcfJ and MEFC::mcfJ obtained in Examples 4 and 5 with promoter replacement and copy number increase of mcfJ and the control strain Coleophoma sp.MEFC009 were inoculated on PDA solid plates and cultured at 25°C. 5-7 days. Pick a small amount of mycelium and use a nucleic acid extractor ( -24) Disintegrate the mycelium, and inoculate the broken mycelium into 50 mL of Coleophoma sp seed culture medium (250 mL Erlenmeyer flask) at 25°C, 220 rpm, and culture on a shaker for 45-48 hours.
  • a nucleic acid extractor -24
  • the HPLC analysis method is: the liquid chromatography column is Agilent C-18 reverse column 883975-902 (4.6 ⁇ 150mm, 5 ⁇ m); the mobile phase is A: 0.05% (volume ratio) trifluoroacetic acid aqueous solution, mobile phase B: 0.05% (Volume ratio) trifluoroacetic acid acetonitrile solution, flow rate is 1mL/min, UV detection wavelength: 210nm, 30°C, total elution time is 25min.
  • FR901379 in the engineering strains MEFC-PgpdAt-mcfJ and MEFC::mcfJ was significantly higher than that of the control strain Coleophoma sp.MEFC009.
  • the production of FR901379 in the engineering strain MEFC-PgpdAt-mcfJ was 1116 mg/L, which was increased compared with the control strain. 241%; the production of FR901379 in the engineered strain MEFC::mcfJ was 1383.5mg/L, which was increased by 268.9% compared with the control strain Coleophoma sp.MEFC009, as shown in Figure 7.
  • mcfJ homologous genes in other strains that can synthesize echinocandins; for example, in the echinocandin B-producing strain Aspergillus nidulans ARTP-7 (strain accession number: CGMCC No. 40073), and ctg12_1653 in the neomocontin B 0 production strain KB01 (Glarea lozoyensis ATCC 74030); the sequence similarity between mcfJ and ecdJ is 49%; the sequence similarity between mcfJ and ctg12_1653 is 60%.
  • the nucleic acid sequence and amino acid sequence of EcdJ are shown in SEQ ID No. 3 and SEQ ID No. 4 respectively; the nucleic acid sequence and amino acid sequence of ctg12_1653 are shown in SEQ ID No. 5 and SEQ ID No. 6 respectively.
  • Example 7 Construction of engineering strain ARTP-7- ⁇ ecdJ with knockout gene ecdJ
  • ecdJ In the biosynthetic gene cluster of echinocandin B, there is the gene ecdJ, whose amino acid sequence is annotated as an unknown functional protein. It is speculated that ecdJ may be related to the synthesis of echinocandin B. In order to verify its function, the gene ecdJ was knocked out.
  • the nucleic acid sequence and amino acid sequence of EcdJ are shown in SEQ ID No. 3 and SEQ ID No. 4 respectively.
  • PCR amplification was performed using primers hph-F (5'-ttcgggatcgcaagcgtaag-3') and hph-R (5'-caattatctttgcgaacccagg-3') to obtain hygromycin of approximately 2.2 kb.
  • Resistance screening fragment hph fuse the hph fragment, the upstream sequence U-ecdJ and the downstream sequence D-ecdJ through fusion PCR, and then use the fusion product as a template and use the nested primer UecdJ-CS-F (5'-aaaaacaacggcgattcttag-3 ') and DecdJ-CS-R (5'-ctgcagctgttaatgtggat-3') were amplified by PCR to amplify the knockout targeting element UecdJ-hph-DecdJ with a size of 4.4kb.
  • A.nidulans ARTP-7 as the starting bacteria, take 1 to 2 mL of sterile physiological saline on a PDA plate, wash the spores with a sterile brush, filter the washed spores with a 300 to 500 mesh filter cloth, collect the spores and suspend them The liquid was counted with a spore counter, and 10 8 CFU/mL spore suspension was inoculated into 50 mL of seed culture medium, cultured on a shaking table at 25°C, 220 rpm for 1 day, and the culture medium and mycelium were poured into a 50 mL sterile centrifuge. tube, centrifuge at 4500 rpm to collect mycelium.
  • the three engineering strains ARTP-7- ⁇ ecdJ and the control strain ARTP-7 were inoculated on PDA solid plates and cultured at 25°C for 5-7 days. Take 1 to 2 mL of sterile physiological saline on a PDA plate, wash the spores with a sterile brush, and filter the washed spores with a 300 to 500 mesh filter cloth. Collect the spore suspension and count it with a spore counter. Increase the size (10 8 CFU/mL) spore suspension was inoculated into 50 mL of seed culture medium, and cultured on a shaker at 25°C, 220 rpm for 2 days.
  • the HPLC analysis method is: the liquid chromatography column is Agilent C-18 reverse column 883975-902 (4.6 ⁇ 150mm, 5 ⁇ m); the mobile phase is A: 0.05% (volume ratio) formic acid aqueous solution, mobile phase B: 0.05% (volume ratio) Ratio) formic acid acetonitrile solution, flow rate is 1mL/min, UV detection wavelength: 210nm, 30°C, total elution time is 25min.
  • PCR amplification was performed using primers ecdJ-F (5'-acaactcatcaatcatcacaATGCACTTCGCAGAGAGCGT-3') and ecdJ-R (5'-caaaattcttcatttatttaCTATACTTGCTTACATAGGC-3') to obtain a size of approximately 2.0kb. Fragments of size ecdJ.
  • ecdJ-F 5'-acaactcatcaatcatcacaATGCACTTCGCAGAGAGCGT-3'
  • ecdJ-R 5'-caaaattcttcatttattttaCTATACTTGCTTACATAGGC-3'
  • PCR amplification was performed using primers PgpdAt-F (5'-ccgtagggatggccataatg-3') and Tpgk-R (5'-attgcagcgcacaagtca-3') to obtain a fragment PgpdAt of approximately 3.6kb. -ecdJ-TpgK.
  • A.nidulans ARTP-7 as the starting bacteria, take 1 to 2 mL of sterile physiological saline on a PDA plate, wash the spores with a sterile brush, filter the washed spores with a 300 to 500 mesh filter cloth, collect the spores and suspend them The liquid was counted with a spore counter, and 10 8 CFU/mL spore suspension was inoculated into 50 mL of seed culture medium, cultured on a shaking table at 25°C, 220 rpm for 1 day, and the culture medium and mycelium were poured into a 50 mL sterile centrifuge. tube, centrifuge at 4500 rpm to collect mycelium.
  • the engineering strain overexpressing ecdJ and the control strain A.nidulans ARTP-7 were inoculated on PDA solid plates and cultured at 25°C for 5-7 days. Take 1 to 2 mL of sterile physiological saline on a PDA plate, wash the spores with a sterile brush, and filter the washed spores with a 300 to 500 mesh filter cloth. Collect the spore suspension and count it with a spore counter. Increase the size (10 8 CFU/mL) spore suspension was inoculated into 50 mL of seeds. Culture medium, 25°C, 220rpm, shaker culture for 2 days.
  • the HPLC analysis method is: the liquid chromatography column is Agilent C-18 reverse column 883975-902 (4.6 ⁇ 150mm, 5 ⁇ m); the mobile phase is A: 0.05% (volume ratio) formic acid aqueous solution, mobile phase B: 0.05% (volume ratio) Ratio) formic acid acetonitrile solution, flow rate is 1mL/min, UV detection wavelength: 210nm, 30°C, total elution time is 25min.
  • ctg12_1653 is abbreviated as 1653
  • the nucleic acid sequence and amino acid sequence of ctg12_1653 are shown in SEQ ID No. 5 and SEQ ID No.
  • the constructed plasmid was introduced into E. coli to increase the copy number.
  • PCR amplification was performed using primers PgpdAf (5'-CCGTAGGGATGGCCATAATG-3') and hphr (5'-CAATTATCTTTGCGAACCCAGG-3'), and the 6 kb overexpression fragment PgpdAt-1653-Tpgk-hph was obtained. .
  • the engineering strain KB01 was inoculated on PDA plates and cultured at 28°C for 4 to 6 days.
  • the mycelium was washed twice with 1M KCl. After pressing dry, place it in a sterile 50mL Erlenmeyer flask, add an appropriate amount of enzymatic hydrolysis solution according to the weight of mycelium (add 10mL of enzymatic hydrolysis solution for every 1g of mycelium), and process at 30°C and 130rpm for 1 to 3 hours.
  • the components of the enzymatic solution are: 0.5-1% wall-lysing enzyme, 0.5-1% helicase and 1M KCl. Filter and sterilize through a 0.22 ⁇ m sterile filter. Filter the above enzymatically hydrolyzed mixture with 500 mesh nylon cloth or lens cleaning paper, and collect the filtrate.
  • Protoplasts were collected by centrifugation at 4000 rpm and 4°C. Wash once with ice-cold STC, re-suspend the protoplasts in the pre-cooled STC, and use STC to adjust the protoplast concentration to 5 ⁇ 10 7 /mL to obtain a protoplast suspension.
  • the engineering strain overexpressing ctg12_1653 and the control strain KB01 were inoculated on PDA solid plates and cultured at 25°C for 5-7 days.
  • Pick a small amount of mycelium and use a nucleic acid extractor ( -24) Disintegrate the mycelium, inoculate the broken mycelium into 50 mL KB01 seed culture medium (250 mL Erlenmeyer flask), and culture on a shaking table at 25°C, 220 rpm for 96-120 hours.
  • the HPLC analysis method is: The HPLC analysis method is: the liquid chromatography column is Agilent C-18 reverse column 883975-902 (4.6 ⁇ 150mm, 5 ⁇ m); the mobile phase is A: aqueous solution, mobile phase B: acetonitrile solution, the flow rate is 1mL /min, UV detection wavelength: 210nm, 30°C, total elution time is 10min. Elution conditions: 30 to 60% mobile phase A.
  • the yield of neumocontin B 0 in the wild-type KB01 strain is 1623 mg/L.
  • the highest yield of compound neumocontin B 0 in the engineered strain KB01::1653 is 2809 mg/L, which is the same as the control strain wild type.
  • the production of KB01 increased by 75% compared to niumocantine B 0 ; in Figure 13, KB01 is the wild-type strain, and KB01::1653 is the KB01 strain overexpressing ctg12_1653.

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Abstract

本发明提供了一种提高棘白菌素类化合物产量的转录因子，所述转录因子在出发菌株中过表达可以提高棘白菌素类化合物的产量；本发明还提供了上述转录因子在制备高产棘白菌素类化合物的基因工程菌株中的应用；还提供了上述基因工程菌株在制备棘白菌素类化合物中的应用。

Description

提高棘白菌素类化合物产量的转录因子及其应用 技术领域
本发明属于基因工程技术领域，涉及一种提高棘白菌素类化合物产量的转录因子及其应用，具体涉及过表达合成途径中的特异性转录调控因子使FR901379、棘白菌素B和纽莫康定B0产量提高的基因工程菌及其构建方法与应用。
背景技术
棘白菌素类药物前体是一类由丝状真菌产生的环脂肽类化合物，该类药物能够选择性地抑制真菌细胞壁中β-1,3葡聚糖合成酶的活性，从而影响真菌细胞壁的合成，导致真菌细胞裂解死亡。由于哺乳动物没有细胞壁，所以该类药物对人体的毒副作用小，安全性高，此外该类药物对耐药菌有效。目前临床上应用的棘白菌素类抗真菌药物包括卡泊芬净、米卡芬净和阿尼芬净，它们的前体化合物分别是纽莫康定B0、FR901379和棘白菌素B，它们的环脂肽骨架都是由非核糖体多肽合酶和脂肪酸或聚酮合酶共同催化合成。
目前棘白菌素类药物前体发酵生产过程中面临着主产物浓度低，副产物多等问题，使得该类药物的生产成本一直居高不下，即使该类药物具有很好的抗真菌效果，但是由于其生产成本高、价格昂贵，一般情况下并不使用。
由转录因子组成的代谢调控***具有全局、动态调控目标代谢途径的能力，在代谢工程中得到广泛的应用。虽然棘白菌素B和纽莫康定B0的生物合成途径已经被解析，其生物合成基因簇中都未能发现途径特异性转录调控因子，因此未有关于基于特异性转录调控因子提高棘白菌素类化合物效价的相关报道。本研究在米卡芬净前体FR901379生物合成基因簇中首次发现了特异性转录调控因子，并通过对转录因子启动子替换以及增加拷贝数两种策略，使得FR901379的产量显著提高，该研究提供了一种有效提高棘白菌素类化合物产量的有效策略。
发明内容
一方面，本发明提供了一种提高棘白菌素类化合物产量的转录因子，所述转录因子的氨基酸序列与SEQ ID No.2或SEQ ID No.4或SEQ ID No.6相比具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的序列同一性。
在一个实施方式中，所述转录因子为转录因子McfJ，所述McfJ的氨基酸序列与SEQ ID No.2相比，具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的序列同一性，优选的，所述McfJ来源于鞘茎点霉属真菌，例如，鞘茎点霉(Coleophoma sp.)MEFC009。优选的，所述McfJ的氨基酸序列如SEQ ID No.2所示。
在一个实施方式中，所述转录因子为转录因子EcdJ，所述EcdJ的氨基酸序列与SEQ ID No.4相比，具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的序列同一性，优选的，所述EcdJ来源于Aspergillus nidulans，例如，保藏编号为CGMCC No.40073的构巢曲霉(Aspergillus nidulans)ARTP-7。优选的，所述EcdJ的氨基酸序列如SEQ ID No.4所示；上述菌株(Aspergillus nidulans)ARTP-7记载在中国专利申请CN114907989A中。
在一个实施方式中，所述转录因子为转录因子ctg12_1653，所述ctg12_1653的氨基酸序列与SEQ ID No.6相比，具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的序列同一性，优选的，所述ctg12_1653来源于Glarea sp.，例如，Glarea lozoyensis,例如，Glarea lozoyensis ATCC 74030。优选的，所述ctg12_1653的氨基酸序列如SEQ ID No.6所示。
另一方面，本发明还提供了包含上述转录因子或其编码基因的生物材料。所述生物材料选自：包含上述转录因子的载体，或者，包含上述转录因子的宿主细胞。
另一方面，本发明还提供了编码上述转录因子的基因。
另一方面，本发明还提供了包含上述基因的载体，或者，包含所述载体的宿主细胞。
在一个实施方式中，所述载体包括克隆载体和表达载体，例如，pET系列载体(如，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系列载体(如pMAL-2c)、pGEX系列载体(如pGEX-4T-2、pGEX-6T-1)、pBAD系列载体(如pBAD-His、pBAD-Myc)、pMBP系列载体(pMBP-P、pMBP-C)、pTYB2、pQE-9、pACYCDuet-1、 pCDFDuet-1、pColADuet-1、pRSFDuet-1、pllP-OmpA、pUC系列载体(如，pUC18、pUC19)，pQE-30、pXH2-1，pXH-43，pTRII，pGSF957。
在一个实施方式中，所述宿主细胞选自大肠杆菌(例如，大肠杆菌DH5α、大肠杆菌BL21(DE3)、Rosetta(DE3)、Codon Plus(DE3)-RIPL、BL21Codon plus(DE3)、Top 10、JM109)、酵母菌(例如，酿酒酵母、毕赤酵母、解酯耶氏酵母)、鞘茎点霉真菌、Aspergillus nidulans、Aspergillus pachycristatus、A.delacroxii、E.rugulosa、E.nidulans或Glarea sp.。
另一方面，本发明还提供了上述转录因子、其编码基因、包含基因的载体、上述宿主细胞、或上述生物材料在制备棘白菌素类化合物中的应用。在一个实施方式中，所述棘白菌素类化合物为FR901379、棘白菌素B或纽莫康定B0。
在一个实施方式中，本发明提供了转录因子McfJ、其编码基因、包含基因的载体、上述宿主细胞、或上述生物材料在制备棘白菌素类化合物中的应用；所述棘白菌素类化合物为FR901379。
在一个实施方式中，本发明提供了转录因子EcdJ、其编码基因、包含基因的载体、上述宿主细胞、或上述生物材料在制备棘白菌素类化合物中的应用；所述棘白菌素类化合物为棘白菌素B。
在一个实施方式中，本发明提供了转录因子ctg12_1653、其编码基因、包含基因的载体、上述宿主细胞、或上述生物材料在制备棘白菌素类化合物中的应用；所述棘白菌素类化合物为纽莫康定B0。
另一方面，本发明还提供了上述转录因子、其编码基因、包含基因的载体、上述宿主细胞、或上述生物材料在制备高产棘白菌素类化合物的基因工程菌株中的应用；在一个实施方式中，所述转录因子为McfJ，所述棘白菌素类化合物为FR901379，所述基因工程菌株的出发菌株为鞘茎点霉属真菌，优选，鞘茎点霉(Coleophoma sp.)MEFC009；在其他的实施方式中，所述转录因子为EcdJ，所述棘白菌素类化合物为棘白菌素B，所述基因工程菌株的出发菌株为Aspergillus nidulans、Aspergillus pachycristatu、Emericella nidulans、A.delacroxii、E.rugulosa或E.nidulans，所述出发菌株能够产生棘白菌素B，优选，Aspergillus nidulans；在其他的实施方式中，所述转录因子为ctg12_1653，所述棘白菌素类化合物为纽莫康定B0，所述基因工程菌株的出发菌株为Glarea sp.，优选，Glarea lozoyensis。
上述制备高产棘白菌素类化合物的基因工程菌株，为在出发菌株中引入上述转录因子或其编码基因；优选的，所述引入为过表达。
另一方面，本发明还提供了一种高产棘白菌素类化合物的基因工程菌株，所述基因工程菌株为在出发菌株中过表达上述转录因子而得到的基因工程菌株。在一个实施方式中，所述转录因子为McfJ，所述棘白菌素类化合物为FR901379，所述基因工程菌株的出发菌株为鞘茎点霉属真菌，优选，鞘茎点霉(Coleophoma sp.)MEFC009；在其他的实施方式中，所述转录因子为EcdJ，所述棘白菌素类化合物为棘白菌素B，所述基因工程菌株的出发菌株为Aspergillus nidulans、Aspergillus pachycristatu、Emericella nidulans、A.delacroxii、E.rugulosa或E.nidulans，优选，Aspergillus nidulans；在其他的实施方式中，所述转录因子为ctg12_1653，所述棘白菌素类化合物为纽莫康定B0，所述基因工程菌株的出发菌株为Glarea sp.，优选，Glarea lozoyensis。
另一方面，本发明还提供了一种制备高产棘白菌素类化合物的基因工程菌株的方法，所述方法包括在出发菌株中过表达上述转录因子从而制备得到所述基因工程菌株的步骤。在一个实施方式中，所述转录因子为McfJ，所述棘白菌素类化合物为FR901379，所述基因工程菌株的出发菌株为鞘茎点霉属真菌，优选，鞘茎点霉(Coleophoma sp.)MEFC009；在其他的实施方式中，所述转录因子为EcdJ，所述棘白菌素类化合物为棘白菌素B，所述基因工程菌株的出发菌株为Aspergillus nidulans、Aspergillus pachycristatu、Emericella nidulans、A.delacroxii、E.rugulosa或E.nidulans，优选，Aspergillus pachycristatu；在其他的实施方式中，所述转录因子为ctg12_1653，所述棘白菌素类化合物为纽莫康定B0，所述基因工程菌株的出发菌株为Glarea sp.，优选，Glarea lozoyensis。
本发明中的“过表达”是指基因工程菌与野生型的出发菌株相比，所述目的基因的表达量或活性或表达水平要高于野生型的出发菌株。在一个实施方式中，上述过表达可以通过引入表达载体来过表达目的基因来实现；其他的实施方式中，上述过表达也可以通过在出发菌株中引入额外的目的基因的拷贝、通过增加目的基因的拷贝数来实现；其他的实施方式中，还可以通过对目的基因的启动子优化来实现，比如，通过将目的基因的原始启动子替换为活性更高的启动子来实现目的基因的过表达。
本发明中的过表达包括通过引入额外的目的基因的拷贝，增加目的基因在细胞中的拷贝数来提高基因的表达量，或者通过对基因的启动子进行替换以提高目的基因的表达量。
在一个实施方式中，所述过表达包括将转录因子的原始启动子替换为强启动子的步骤；优选的，所述强启动子为启动子PgpdAt。
在另一个实施方式中，所述过表达包括在出发菌株中引入额外的转录因子的拷贝的步骤。
在一个实施方式中，所述的“引入”包括将目的基因构建到外源表达载体上，将外源表达载体转入到 出发菌株中以过表达转录因子；优选的，所述外源表达载体包括pXH2-1，pXH43，pTRII，pGSF957。
在另一个实施方式中，所述的“引入”包括将目的基因***到出发菌株的基因组中；优选的，所述***到基因组中可以采用同源重组双交换的方法；在一个实施方式中，可以采用将目的基因以及同源臂***到载体上，然后将载体转入到出发菌株中，利用同源臂与基因组发生同源重组双交换从而将目的基因***到合适的基因组的位置；在其他的实施方式中，还可以采用基因编辑的方式，例如，利用CRISPR/Cas***在期望的基因组位点上进行切割，同时将目的基因作为外源供体***到切割位点上。
在优选的实施方式中，将额外的转录因子拷贝***到出发菌株的基因组时，所述额外的转录因子拷贝在强启动子的作用下进行表达；优选的，所述强启动子为启动子PgpdAt。
在其他的实施方式中，所述强启动子优选为启动子PgpdAt，PcitA，PgpdA，PtrpC，Pgpk，Pmpgd。上述启动子均是本领域已知的启动子，如期刊文献Huang X et al.Cloning,characterization and application of a native glyceraldehyde-3-phosphate dehydrogenase promoter for Aspergillus terreus.J Ind Microbiol Biotechnol 2014,41:585–592.公开了PgpdAt。以及期刊文献Dave K et al.Utility of Aspergillus niger citrate synthase promoter or heterologous expression.J Biotechnol 2011,155:173–177公开了PcitA。文献Kim JG et al.Genetic transformation of Monascus purpureus DSM1379.Biotechnol Lett,2003,25:1509–1514公开了Pmgpd和Ptrp(即PtrpC)。文献Punt PJ,et al.Functional elements in the promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase.Gene,1990,93:101–109公开了PgpdA。文献Nara,F.et al.Cloning and sequencing of the 3-phosphoglycerate kinase(PGK)gene from Penicillium citrinum and its application to heterologous gene expression.Curr Genet,1993,23(2):132-140公开了Ppgk。
另一方面，本发明还提供了上述高产棘白菌素类化合物的基因工程菌株在生产棘白菌素类化合物中的应用。所述高产棘白菌素类化合物的基因工程菌株为在出发菌株中过表达上述转录因子而得到的基因工程菌株；在一个实施方式中，所述转录因子为McfJ，所述棘白菌素类化合物为FR901379，所述基因工程菌株的出发菌株为鞘茎点霉属真菌，优选，鞘茎点霉(Coleophoma sp.)MEFC009；在其他的实施方式中，所述转录因子为EcdJ，所述棘白菌素类化合物为棘白菌素B，所述基因工程菌株的出发菌株为Aspergillus nidulans、Aspergillus pachycristatu、Emericella nidulans、A.delacroxii、E.rugulosa或E.nidulans，优选，Aspergillus nidulans；在其他的实施方式中，所述转录因子为ctg12_1653，所述棘白菌素类化合物为纽莫康定B0，所述基因工程菌株的出发菌株为Glarea sp.，优选，Glarea lozoyensis。
另一方面，本发明还提供了一种制备棘白菌素类化合物的方法，所述方法包括利用上述基因工程菌株进行发酵的步骤；任选的，所述方法还包括分离/纯化所述棘白菌素类化合物的步骤。
在一个实施方式中，所述棘白菌素类化合物为FR901379，所述基因工程菌株为在出发菌株中过表达上述转录因子而得到的基因工程菌株，所述转录因子为McfJ，所述基因工程菌株的出发菌株为鞘茎点霉属真菌，优选，鞘茎点霉(Coleophoma sp.)MEFC009。
在其他的实施方式中，所述棘白菌素类化合物为棘白菌素B，所述基因工程菌株为在出发菌株中过表达上述转录因子而得到的基因工程菌株，所述转录因子为EcdJ，所述基因工程菌株的出发菌株为Aspergillus nidulans、Aspergillus pachycristatu、Emericella nidulans、A.delacroxii、E.rugulosa或E.nidulans，优选，Aspergillus nidulans。
在其他的实施方式中，所述棘白菌素类化合物为纽莫康定B0，所述基因工程菌株为在出发菌株中过表达上述转录因子而得到的基因工程菌株，所述转录因子为ctg12_1653，所述基因工程菌株的出发菌株为Glarea sp.，优选，Glarea lozoyensis。
另一方面，本发明还提供了一种提高目的菌株中棘白菌素类化合物产量的方法，所述方法包括在目的菌株中过表达上述转录因子的步骤。在一个实施方式中，所述棘白菌素类化合物为FR901379，所述转录因子为McfJ，所述目的菌株为鞘茎点霉属真菌，优选，鞘茎点霉(Coleophoma sp.)MEFC009。在其他的实施方式中，所述棘白菌素类化合物为棘白菌素B，所述转录因子为EcdJ，所述目的菌株为Aspergillus nidulans、Aspergillus pachycristatu、Emericella nidulans、A.delacroxii、E.rugulosa或E.nidulans，优选，Aspergillus nidulans。在其他的实施方式中，所述棘白菌素类化合物为纽莫康定B0，所述转录因子为ctg12_1653，所述目的菌株为Glarea sp.，优选，Glarea lozoyensi。
附图说明
图1为敲除mcfJ基因所获转化子的基因组PCR验证结果；其中2#，5#和8#：基因mcfJ缺失的转化子，WT：对照菌株Coleophoma sp.MEFC009，M：1kb Marker。
图2为基因mcfJ缺失菌株发酵产物HPLC分析结果；MEFC009：MEFC--ΔmcfJ为mcfJ缺失菌株；对照菌株Coleophoma sp.MEFC009；1：FR901379。
图3为FR901379生物合成基因转录水平分析；其中MEFC-ΔmcfJ为基因mcfJ缺失菌株，MEFC009为对照菌株Coleophoma sp.MEFC009，8763_g：肌动蛋白编码基因，其他mcf基因为负责FR901379合成的基因。
图4为转录调控因子mcfJ启动子替换所获得的转化子基因组PCR验证；其中1-8为转化子，WT-1为Coleophoma sp.MEFC-Δku80，M：1kb Marker。
图5为增加转录调控因子mcfJ拷贝数的工程菌株MEFC::mcfJ与Coleophoma sp.MEFC009中基因mcfJ转录水平分析，1,2：对照菌株Coleophoma sp.MEFC009；3,4：工程菌株MEFC::mcfJ；8763_g：肌动蛋白编码基因。
图6为转录调控因子McfJ启动子替换和增加拷贝数的工程菌株MEFC-PgpdAt-mcfJ和MEFC::mcfJ及对照菌株Coleophoma sp.MEFC009发酵产物HPLC分析结果；其中1为FR901379。
图7为过表达转录调控因子McfJ的工程菌株MEFC-PgpdAt-mcfJ、MEFC::mcfJ和Coleophoma sp.MEFC009发酵液中FR901379产量分析。
图8敲除ecdJ基因所获转化子的基因组PCR验证结果；其中8#和16#是基因ecdJ缺失的转化子，C2为野生型对照，C3为阳性对照。
图9基因ecdJ缺失菌株A.nidulans-ΔecdJ发酵产物HPLC分析结果；其中ARTP-7-ΔecdJ为基因ecdJ缺失菌株，ARTP-7为对照菌株。
图10过表达转录调控因子EcdJ所获得的转化子基因组PCR验证；其中1-4号为转化子，C1为阴性对照，C2为阳性对照。
图11过表达转录调控因子EcdJ的工程菌株和A.nidulans.ARTP-7发酵液中棘白菌素B产量分析；其中，ARTP-7为野生型对照，ARTP-7::ecdJ为ecdJ过表达菌株。
图12过表达转录调控因子ctg12_1653所获得的转化子基因组PCR验证；1-4：转化子基因组。
图13过表达转录调控因子ctg12_1653的工程菌株和对照菌株KB01发酵液中纽莫康定B0产量分析。
具体实施方式
下面结合具体实施例对本发明做进一步说明，但本发明不受实施例的限制。以下实施例中所用材料、试剂、仪器和方法，未经特殊说明，均为本领域中的常规材料、试剂、仪器和方法，均可通过商业渠道获得。
本实施方式中涉及的McfJ、EcdJ和ctg12_1653的核酸序列和氨基酸序列如下表所示：


本发明中PCR片段纯化采用OMEGA公司DNA片段回收Cycle-Pure Kit试剂盒(D6492-01)；PCR高保真酶和一步克隆酶Ultra One Step Cloning Kit购自南京Vazyme公司；限制性内切酶购自美国赛默飞公司；RNA提取试剂盒和RNA反转录试剂盒均购自TaKaRa。
MEFC009的种子培养基：15g/L可溶性淀粉，10g/L蔗糖，5g/L棉籽饼粉，10g/L蛋白胨，1g/L KH2PO4，2g/L CaCO3。
MEFC009的发酵培养基：30g/L玉米淀粉，30g/L蛋白胨，6g/L(NH4)2SO4，1g/L KH2PO4，0.3g/L FeSO4·7H2O，0.01g/L ZnSO4·7H2O，2g/L CaCO3。
构巢曲霉(Aspergillus nidulans)ARTP-7的种子培养基：5～30g/L棉籽饼粉，5～20g/L葡萄糖，5～10g/L甘油，用NaOH调节至pH 5.0～7.0。
构巢曲霉(Aspergillus nidulans)ARTP-7的发酵培养基：60～150g/L甘露醇，15～50g/L花生油，5～20g/L甘油，5～20g/L黄豆饼粉，5～20g/L蛋白胨，0.01～0.07g/L FeSO4·7H2O，5～10g/L K2HPO4，0.2～1g/L MgSO4·7H2O，0.1～0.8g/L MnSO4·H2O，0.3～0.8g/L CuSO4·5H2O，0.1～0.5g/L CaCl2。
KB01的种子培养基：一水葡萄糖20～50g，黄豆饼粉10～30g，棉籽饼粉5～20g，玉米浆干粉5～20g，KH2PO4 0.05～0.2g，微量元素群(1mL/100mL)。(微量元素群：FeSO4·7H2O 1g，MnSO4·H2O 1g，ZnSO4·7H2O 0.2g，CaCl2·2H2O 0.1g，H3BO3 0.056g，CuCl2·2H2O 0.025g，(NH4)6Mo7O24·4H2O 0.01g)，pH 5.3～5.5。
KB01的发酵培养基：山梨醇100～150g，一水葡萄糖1～10g，棉籽饼粉5～20g，麸质粉20～50g，K2HPO4·3H2O 1～5g，(NH4)2SO4 0.2～2g，NaNO3 0.2～2g，FeSO4·7H2O 0.1～1g，L-脯氨酸5～30g，L-苏氨酸1～5g，pH 6.8。
STC：1M山梨醇，50mM Tris-HCl(pH 8.0)，50mM CaCl2。
PSTC：40％PEG4000，1M山梨醇，50mM Tris-HCl(pH 8.0)，50mM CaCl2。
顶层琼脂：PDB、1M山梨醇和4g/L琼脂糖，灭菌后45-48℃保温。
再生筛选培养基平板PDA-SH：PDA平板、1M山梨醇和100mg/L潮霉素B。
筛选培养基PDA-H：PDA平板和100mg/L潮霉素B。
质粒pXH2-1记载在Xuenian Huang，Xuefeng Lu，Jian-Jun Li.Cloning,characterization and application of a glyceraldehyde-3-phosphate dehydrogenase promoter from Aspergillus terreus,J Ind Microbiol Biotechnol(2014)41:585–592。
质粒PU19-ZX由本实验室自主构建。
本实施方式中，所利用的出发菌株为鞘茎点霉属真菌，鞘茎点霉(Coleophoma sp.)MEFC009，上述菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏编号为CGMCC No.21058，保藏日期为2020年11月18日，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，电话：010-64807355。Glarea lozoyensis ATCC 74030均购自美国标准生物品收藏中心(ATCC)，本文中，又将Glarea lozoyensis ATCC 74030称之为KB01。构巢曲霉(Aspergillus nidulans)ARTP-7是以(Aspergillus nidulans)AN01为出发菌株，通过ARTP诱变获得，所述菌株保藏编号为CGMCC No.40073，所述菌株于2022年01月29日保藏于中国微生物菌种保藏管理委员会普通微生物中心；上述菌株(Aspergillus nidulans)ARTP-7记载在中国专利申请CN114907989A中。
实施例1.构建敲除基因mcfJ的工程菌株MEFC-ΔmcfJ
在FR901379生物合成基因簇中，存在基因mcfJ，其氨基酸序列被注释为未知功能蛋白，通过转录组数据分析发现mcfJ与其他负责FR901379合成的基因转录水平相当，猜测mcfJ可能与FR901379的合成相关。为了对其功能进行验证，对基因mcfJ进行了敲除。McfJ的核酸序列和氨基酸序列分别如SEQ ID No.1和SEQ ID No.2所示。
以野生型Coleophoma sp.MEFC009的基因组为模板，采用pfu DNA聚合酶(Fermentas,Catalog No.:EP0501)进行PCR扩增，用引物UmcfJ-F(5’-ccggtggcttgaaagatttc-3’)和UmcfJ-R(5’-ctttacgcttgcgatcccgaaGTTAACTACGGACATACCT-3’)可以扩增获得大小约为1.3kb的mcfJ的上游序列U-mcfJ，用引物DmcfJ-F(5’-cctgggttcgcaaagataattgCGTCAACATCGGTGATACCC-3’)和DmcfJ-R(5’-aggaggactcgaaatcaaag-3’)可以扩增获得大小为1.4kb的mcfJ的下游序列D-mcfJ。以质粒pXH2-1为模板，用引物hph-F(5’-ttcgggatcgcaagcgtaaag-3’)和hph-R(5’-caattatctttgcgaacccagg-3’)进行PCR扩增，获得大小约为2.2kb的潮霉素抗性筛选片段hph；通过融合PCR将hph片段、上游序列U-mcfJ和下游序列D-mcfJ进行融合，再以融合产物为模板，用巢式引物UmcfJ-CS-F(5’-gcctagcctggccatatgca-3’)和DmcfJ-CS-R(5’-ctttatccggacctacagc-3’)通过PCR扩增出大小为4.6kb的敲除打靶元件UmcfJ-hph-DmcfJ。
以Coleophoma sp.MEFC009为出发菌，首先从PDA平板上取少量菌丝，用手持匀浆器破碎，取1mL种子液接种到50mL的种子培养基，于250mL的三角瓶中，220rpm，25℃进行摇床培养。2天后，离心收集菌丝。5000rpm，4℃，5min。将菌丝体再次用匀浆器破碎，取0.5mL-2mL种子液接种到50mL的种子培养基，相同的条件下培养1天，将培养基与菌丝体一起倒入50mL的无菌离心管中，5000rpm，离心收集菌丝。用0.6M MgSO4对菌丝体进行清洗2次。将菌丝体清洗至白色，称取1g菌丝，加入10mL酶解液，30℃，100rpm处理1-4h。酶解液成分为：1％纤维素酶，0.6％溶壁酶、0.6％蜗牛酶和0.6M MgSO4，经由0.22μm的无菌过滤器过滤除菌。将上述原生质体反应液通过无菌神奇滤布进行过滤。5000rpm，4℃离心收集原生质体。用冰预冷的STC，洗涤一次，把原生质体重悬于预冷的STC中，并用STC将原生质体浓度调整为5×107个/mL，得到原生质体悬液。
向140μL上述原生质体悬液中加入10μL的UmcfJ-hph-DmcfJ片段，再加入50μL的PSTC，轻轻混匀，冰浴30min。加入1mL PSTC，混匀后室温放置20min；然后与10mL顶层琼脂混合后倾注于3块再生筛选培养基平板PDA-SH上，在30℃、黑暗条件下培养5-7天，得到转化子。
从转化筛选平板上挑取具有潮霉素抗性转化子转移至PDA-H上，在25℃培养5-7天进行传代培养，连续传代3代。选取稳定传代的3个转化子2#，5#，8#进行单孢分离纯化，并提取单孢分离之后的转化子基因组。利用外部引物UmcfJ-F(5’-ccggtggcttgaaagatttc-3’)和DmcfJ-R(5’-aggaggactcgaaatcaaag-3’)对转化子基因组进行PCR验证，能扩增出大小约为4.9kb的条带的为阳性转化子，而Coleophoma sp.MEFC009只能扩增出大小约为3.4kb的条带，图1结果表明在2#，5#，8#转化子为阳性转化子，说明在基因mcfJ位置发生了同源重组，整合了外源片段UmcfJ-hph-DmcfJ，将阳性菌株定义为MEFC-ΔmcfJ。
实施例2.工程菌株MEFC-ΔmcfJ发酵验证
将3株工程菌株Coleophoma sp.-ΔmcfJ-2#，5#，8#和对照菌株Coleophoma sp.MEFC009接种于PDA固体平板上，25℃培养5-7天。挑取少量的菌丝，利用核酸提取仪(-24)对菌丝进行破碎，将破碎后的菌丝接种于50mL Coleophoma sp.的种子培养基(250mL三角瓶)，25℃，220rpm，摇床培养45-48h。将上述培养的种子液取5mL到50mL Coleophoma sp.的发酵培养基(250mL三角瓶)，25℃，220rpm，摇床培养8天，每个菌株设置3个平行。从每一瓶发酵液中取1mL，加入等体积的甲醇，超声萃取1h，离心，取上清。用0.22μm的有机滤器过滤处理样品，并通过HPLC对处理后的样品进行分析。
HPLC分析方法为：液相色谱柱为Agilent C-18反向柱883975-902(4.6×150mm，5μm)；流动相为A：0.05％(体积比)三氟乙酸水溶液，流动相B：0.05％(体积比)三氟乙酸乙腈溶液，流速为1mL/min，紫外检测波长：210nm，30℃，总洗脱时间为37min。梯度洗脱条件：0-5min，流动相B占流动相的体积由5％线性上升到24％，5-35min，流动相B占流动相的体积由24％线性上升到62％，35-37min，流动相B占流动相的体积由62％线性上升到100％。结果如图2所示；与出发菌株相比，MEFC-ΔmcfJ中的FR901379(化合物1)及其中间体完全消失，说明基因mcfJ与FR901379的合成相关。
由于mcfJ应该不是参与FR901379结构形成的基因，故猜测mcfJ可能是转运蛋白或转录调控因子，但是由于FR901379基本上位于细胞内，所以不涉及转运，所以猜测mcfJ很大可能性是转录调控因子。
实施例3.FR901379生物合成相关基因转录水平分析
3.1 RNA的提取
根据以上结果，猜测mcfJ很大可能性是转录调控因子。为了验证该假设，分别提取野生型棘腔孢霉Coleophoma sp.MEFC009和mcfJ缺失菌株中的RNA。1)将新鲜的菌体置于研钵中，加入液氮进行研磨，直至磨成粉末状。将50-100mg菌体转移至灭菌的1.5mL的EP管中，加入450μL Buffer RL(提前加入50×DTT)，用移液器反复吹吸，直至无明显沉淀。2)将裂解液12,000rpm，4℃离心5分钟。3)将上清液小心吸取到新的1.5mL灭菌EP管中。4)加入样品裂解步骤中上清液或混合液1/2体积的无水乙醇，使用移液枪将溶液混合均匀。5)立即将混合液(含沉淀)全部转入到RNA旋转柱中。6)12,000rpm，离心1分钟，弃滤液。将RNA旋转柱放回到2mL收集管中。7)将500μL的Buffer RWA加入至RNA旋转柱中，12,000rpm离心30秒钟，弃滤液。8)将600μL的Buffer RWB加入至RNA旋转柱中，12,000rpm离心30秒钟，弃滤液。9)向RNA旋转柱膜中央加入50μL DNase I反应液，室温静置15分钟。10)将600μL的Buffer RWB加入至RNA旋转柱中，12,000rpm离心30秒钟，弃滤液。11)将RNA旋转柱重新安置于2mL收集管上，12,000rpm离心2分钟。12)将RNA旋转柱安置于1.5mL的无RNA酶的收集管上，在RNA旋转柱膜中央处加入50μL的0.1％DEPC处理水，室温静置5分钟。13)12,000rpm离心2分钟洗脱RNA。
3.2将RNA反转录成cDNA
(1)首先对3.1中提取的RNA残留的DNA进行消化，反应体系如下：5×gDNA Eraser缓冲液2.0μL，gDNA Eraser 1.0μL，总RNA 1.0μg，用无RNA酶的水将反应体系补足至10μL，42℃，放置5min。
(2)反转录：在(1)中的反应液中加入5×PrimerScript II缓冲液4.0μL，RNA酶抑制剂0.5μL，反转录酶1.0μL，RT Primer Mix 1μL，用无RNA酶的水将反应体系补足至20μL，37℃，放置15min，然后85℃，放置5s。
3.3 FR901379合成相关基因转录水平分析
分别在负责FR901379合成的基因的编码区设计引物：mcfKCDS-F(5’-cgatgttgacgaccaattcag-3’)/mcfKCDS-R(5’-tcccgaaaggtgctttcgtt-3’)，mcfICDS-F(5’-cgtcgccaaaatggtgtcaa-3’)/mcfICDS-R(5’-acagcgacaactttgtcctc-3’)，mcfNCDS-F(5’-gtctgttgtaagtcgttgcaga-3’)/mcfNCDS-R(5’-ttacgaagtgtgcccacagt-3’)，mcfECDS-F(5’-atcgttgggttgatgagagtg-3’)/mcfECDS-R(5’-aagggttccaaagtctgagt-3’)，mcfBCDS-F(5’-cctgatcaaaatgctctgcg-3’)/mcfBCDS-R(5’-cgccactggcagtacgtata-3’)，mcfOCDS-F(5’-ccagctcatagaaactcccg-3’)/mcfOCDS-R(5’-gggtcgtctttcgtgatcga-3’)，mcfCCDS-F(5’-tccctggtgcattcttcttct-3’)/mcfCCDS-R(5’-agcaagcaggttagctttgt-3’)，mcfDCDS-F(5’-tttaaatggcggggttccaa-3’)/mcfDCDS-R(5’-ggcgtccttaccaacagtatct-3’)，mcfFCDS-F(5’-gcaccaagtaaaccagcatt-3’)/mcfFCDS-R(5’-agcagtccaatgtccatacc-3’)，mcfHCDS-F(5’-ttggtaatgcagcagcatgt-3’)/mcfHCDS-R(5’-ccaatacgtcatctaaagcgg-3’)，mcfMCDS-F(5’-attccctgcaaaaggacaacc-3’)/mcfMCDS-R(5’-cgtcgtcgatggctcgaaata-3’)，mcfGCDS-F(5’-ctcctagctgtacgaacagaac-3’)/mcfGCDS-R(5’-cctcggactcggtaatgaaat-3’)，mcfLCDS-F(5’-ttgttaggtcgaagtacagcg-3’)/mcfLCDS-R(5’-ctcaggtcgaagctcatcac-3’)和mcfACDS-F(5’-ttcgaacctcggagaatcgag-3’)/mcfACDS-R (5’-gctggtcacttccagactaca’)。以野生型棘腔孢霉Coleophoma sp.MEFC009和mcfJ缺失菌株的cDNA作为模板，通过PCR扩增目的片段，同时以肌动蛋白编码基因8763_g作为对照。通过DNA凝胶电泳对片段进行分析。结果如图3所示，从PCR电泳结果可以看出，当mcfJ缺失后，负责FR901379合成的基因都不再转录，而肌动蛋白编码基因8763_g可以正常转录。说明基因mcfJ为FR901379合成基因簇中的特异性转录调控因子，当mcfJ缺失后会影响其他基因的表达。
实施例4.转录调控因子McfJ启动子替换重组菌株的构建
4.1转录调控因子McfJ启动子替换表达盒的构建
以Coleophoma sp.MEFC009的基因组为模板，利用引物UMcfJ-2F(5’-tacaagcactaatgtaatcg-3’)和UMcfJ-2R(5’-tttacgcttgcgatcccgaatagcaggaatccttatataa-3’)进行PCR扩增，获得大小约为1.2kb大小的片段UMcfJ-2，利用引物DMcfJ-2F(5’-caactcatcaatcatcacaacATGCCTATGCCTATGTCTAC-3’)和DMcfJ-2R(5’-ACATGCCGTGCTGGCGACAT-3’)进行PCR扩增，获得大小约为1.2kb大小的片段DMcfJ-2，以质粒pXH2-1为模板，利用引物hph-F(5’-ttcgggatcgcaagcgtaaag-3’)和hph-R(5’-caattatctttgcgaacccagg-3’)进行PCR扩增，获得大小约为2.2kb的潮霉素抗性筛选片段hph，以质粒pXH2-1为模板，利用引物PgpdAt-F(5’-gttacactctgggaggatcc-3’)和PgpdAt-R(5’-gttgtgatgattgatgagttg-3’)进行PCR扩增，获得大小约为0.75kb的强启动子元件PgpdAt。通过融合PCR的方法，将片段UMcfJ-2、hph、PgpdAt和DMcfJ-2融合起来，以融合产物作为模板，以巢式引物UMcfJ-2CS-F(5’-aatttccctgtaatacacatt-3’)和DMcfJ-2CS-R(5’-ACATGCCGTGCTGGCGACAT-3’)进行PCR扩增，获得大小约为5.1kb的打靶元件UMcfJ-2-hph-PgpdAt-DMcfJ-2。
4.2转录调控因子McfJ启动子替换重组菌株的构建
以Coleophoma sp.MEFC009-Δku80为出发菌，首先从PDA平板上取少量菌丝，用手持匀浆器破碎，取1mL种子液接种到50mL的种子培养基，于250mL的三角瓶中，220rpm，25℃进行摇床培养。2天后，离心收集菌丝。5000rpm，4℃，5min。将菌丝体再次用匀浆器破碎，取0.5mL-2mL种子液接种到50mL的种子培养基，相同的条件下培养1天，将培养基与菌丝体一起倒入50mL的无菌离心管中，5000rpm，离心收集菌丝。用0.6M MgSO4对菌丝体进行清洗2次。将菌丝体清洗至白色，称取1g菌丝，加入10mL酶解液，30℃，100rpm处理1-4h。酶解液成分为：1％纤维素酶，0.6％溶壁酶、0.6％蜗牛酶和0.6M MgSO4，经由0.22μm的无菌过滤器过滤除菌。将上述原生质体反应液通过无菌神奇滤布进行过滤。5000rpm，4℃离心收集原生质体。用冰预冷的STC，洗涤一次，把原生质体重悬于预冷的STC中，并用STC将原生质体浓度调整为5×107个/mL，得到原生质体悬液。
向140μL上述原生质体悬液中加入UMcfJ-2-hph-PgpdAt-DMcfJ-2片段，再加入50μL的PSTC，轻轻混匀，冰浴30min。加入1mL PSTC，混匀后室温放置20min；然后与10mL顶层琼脂混合后倾注于3块再生筛选培养基平板PDA-SH上，在30℃、黑暗条件下培养5-7天，得到转化子。
从转化筛选平板上挑取具有潮霉素抗性转化子转移至PDA-H上，在25℃培养5-7天进行传代培养，连续传代3次。选取8个转化子进行分离纯化，对纯化后的转化子基因组利用引物UMcfJ-2F(5’-tacaagcactaatgtaatcg-3’)和DMcfJ-2R(5’-ACATGCCGTGCTGGCGACAT-3’)进行PCR验证，能扩增出大小约为5.3kb的条带为阳性转化子，而对照菌株只能扩增出大小为2.4kb大小的条带，如图4所示；除了1#转化子外，其他转化子的基因mcfJ启动子都被PgpdAt所替换，将这些转化子命名为MEFC-PgpdAt-mcfJ。
实施例5.转录调控因子McfJ拷贝数增加重组菌株的构建
以Coleophoma sp.MEFC009的基因组为模板，利用引物PMcfJ-F(5’-taatacatcatttcattcat-3’)和TMcfJ-R(5’-cggcctagaaggtcgatcgc-3’)进行PCR扩增，获得大小约为3.5kb大小的片段P-McfJ-T。本实施方式中，并没有对McfJ的启动子进行替换，只是增加了McfJ的拷贝数。
以Coleophoma sp.MEFC009为出发菌，首先从PDA平板上取少量菌丝，用手持匀浆器破碎，取1mL种子液接种到50mL的种子培养基，于250mL的三角瓶中，220rpm，25℃进行摇床培养。2天后，离心收集菌丝。5000rpm，4℃，5min。将菌丝体再次用匀浆器破碎，取0.5mL-2mL种子液接种到50mL的种子培养基，相同的条件下培养1天，将培养基与菌丝体一起倒入50mL的无菌离心管中，5000rpm，离心收集菌丝。用0.6M MgSO4对菌丝体进行清洗2次。将菌丝体清洗至白色，称取1g菌丝，加入10mL酶解液，30℃，100rpm处理1-4h。酶解液成分为：1％纤维素酶，0.6％溶壁酶、0.6％蜗牛酶和0.6M MgSO4，经由0.22μm的无菌过滤器过滤除菌。将上述原生质体反应液通过无菌神奇滤布进行过滤。5000rpm，4℃离心收集原生质体。用冰预冷的STC，洗涤一次，把原生质体重悬于预冷的STC中，并用STC将原生质体浓度调整为5×107个/mL，得到原生质体悬液。
向200μL上述原生质体悬液中加入P-McfJ-T片段和hph片段，再加入50μL的PSTC，轻轻混匀，冰浴30min。加入1mL PSTC，混匀后室温放置20min；然后与10mL顶层琼脂混合后倾注于3块再生 筛选培养基平板PDA-SH上，在30℃、黑暗条件下培养5-7天，得到转化子。
从转化筛选平板上挑取具有潮霉素抗性转化子转移至PDA-H上，在25℃培养5-7天进行传代培养，连续传代3次。选取转化子在发酵培养基中培养1.5天，提取RNA，将其RNA反转录成cDNA，以野生型棘腔孢霉Coleophoma sp.MEFC009作为对照。分别以野生型棘腔孢霉MEFC009和过表达McfJ的突变株MEFC::mcfJ的cDNA作为模板，利用引物McfJCDS-F(5’-atgacgatagtatagagatggtc-3’)和McfJCDS-R(5’-ggagctacacagtgaccttc-3’)，通过PCR进行扩增，同时以肌动蛋白编码基因8503_g作为对照。结果如图5所示，当以工程菌株MEFC::mcfJ的cDNA作为模板时，扩增的条带浓度远高于对照组，说明工程菌株MEFC::mcfJ中基因mcfJ的拷贝数要高于对照菌株Coleophoma sp.MEFC009。
实施例6.过表达转录调控因子McfJ重组菌株的发酵验证
将实施例4和实施例5中得到的mcfJ的启动子替换和拷贝数增加的工程菌株MEFC-PgpdAt-mcfJ和MEFC::mcfJ以及对照菌株Coleophoma sp.MEFC009接种于PDA固体平板上，25℃培养5-7天。挑取少量的菌丝，利用核酸提取仪(-24)对菌丝进行破碎，将破碎后的菌丝接种于50mL Coleophoma sp的种子培养基(250mL三角瓶)，25℃，220rpm，摇床培养45-48h。将上述培养的种子液取5mL到Coleophoma sp.的发酵培养基，25℃，220rpm，摇床培养8天，每个菌株设置3个平行。从每一瓶发酵液中取1mL，加入等体积的甲醇，超声萃取1h，离心，取上清。用0.22μm的有机滤器过滤处理样品，并通过HPLC对处理后的样品进行分析。
HPLC分析方法为：液相色谱柱为Agilent C-18反向柱883975-902(4.6×150mm，5μm)；流动相为A：0.05％(体积比)三氟乙酸水溶液，流动相B：0.05％(体积比)三氟乙酸乙腈溶液，流速为1mL/min，紫外检测波长：210nm，30℃，总洗脱时间为25min。梯度洗脱条件：0-5min，流动相B占流动相的体积由5％线性上升到40％，5-20min，流动相B占流动相的体积由40％线性上升到62％，20-25min，流动相B占流动相的体积由62％线性上升到100％。HPLC分析结果如图6所示，从中可以看出，与野生型Coleophoma sp.MEFC009相比，工程菌株MEFC-PgpdAt-mcfJ和MEFC::mcfJ中化合物FR901379的产量显著提高。工程菌株MEFC-PgpdAt-mcfJ和MEFC::mcfJ中FR901379的产量明显高于对照菌株Coleophoma sp.MEFC009，其中工程菌株MEFC-PgpdAt-mcfJ中FR901379的产量为1116mg/L，与对照菌株相比提高了241％；工程菌株MEFC::mcfJ中FR901379的产量为1383.5mg/L，与对照菌株Coleophoma sp.MEFC009相比提高了268.9％，如图7所示。
我们在其他能够合成棘白菌素类化合物的菌株中，同样找到了mcfJ的同源基因；例如，在棘白菌素B生产菌株构巢曲霉(Aspergillus nidulans)ARTP-7(菌株保藏编号为CGMCC No.40073)中的ecdJ，以及纽莫康定B0生产菌株KB01(Glarea lozoyensis ATCC 74030)中的ctg12_1653；mcfJ与ecdJ的序列相似性是49％；mcfJ与ctg12_1653的序列相似性是60％。EcdJ的核酸序列和氨基酸序列分别如SEQ ID No.3和SEQ ID No.4所示；ctg12_1653的核酸序列和氨基酸序列分别如SEQ ID No.5和SEQ ID No.6所示。
我们在A.nidulans ARTP-7(菌株保藏编号为CGMCC No.40073)和KB01中分别过表达了EcdJ和ctg12_1653，其同样能够提高棘白菌素B和纽莫康定B0的产量，具体实施方式如下：
实施例7.构建敲除基因ecdJ的工程菌株ARTP-7-ΔecdJ
在棘白菌素B的生物合成基因簇中，存在基因ecdJ，其氨基酸序列被注释为未知功能蛋白，猜测ecdJ可能与棘白菌素B的合成相关。为了对其功能进行验证，对基因ecdJ进行了敲除。EcdJ的核酸序列和氨基酸序列分别如SEQ ID No.3和SEQ ID No.4所示。
以A.nidulans ARTP-7的基因组为模板，采用pfu DNA聚合酶(Fermentas,Catalog No.:EP0501)进行PCR扩增，用引物UecdJ-F(5’-ctcggtctttatagtgcgtg-3’)和UecdJ-R(5’-tttacgcttgcgatcccgaaGAGGTCAAAGTTTAGAAAAT-3’)可以扩增获得大小约为1.2kb的ecdJ的上游序列U-ecdJ，用引物DecdJ-F(5’-tgggttcgcaaagataattgGGGCAAGCAGTCCATCGCCC-3’)和DecdJ-R(5’-atgaggatatgactgctctgg-3’)可以扩增获得大小为1.2kb的ecdJ的下游序列D-mcfJ。以质粒pXH2-1为模板，用引物hph-F(5’-ttcgggatcgcaagcgtaaag-3’)和hph-R(5’-caattatctttgcgaacccagg-3’)进行PCR扩增，获得大小约为2.2kb的潮霉素抗性筛选片段hph；通过融合PCR将hph片段、上游序列U-ecdJ和下游序列D-ecdJ进行融合，再以融合产物为模板，用巢式引物UecdJ-CS-F(5’-aaaaacaacggcgattcttag-3’)和DecdJ-CS-R(5’-ctgcagctgttaatgtggat-3’)通过PCR扩增出大小为4.4kb的敲除打靶元件UecdJ-hph-DecdJ。
以A.nidulans ARTP-7为出发菌，取1～2mL无菌生理盐水于PDA平板中，用无菌小毛笔刷洗下孢子，将洗下的孢子用300～500目滤布过滤，收集孢子悬浮液并用孢子计数仪计数，108CFU/mL孢子悬浮液接种量于50mL的种子培养基，25℃，220rpm，摇床培养1天，将培养基与菌丝体一起倒入50mL的无菌离心管中，4500rpm，离心收集菌丝。用0.6M MgCl2对菌丝体进行清洗2次。将菌丝体清洗至白色，称取1g菌丝，加入15mL酶解液，30℃，130rpm处理2-3h。酶解液成分为：1％溶壁酶、0.6％蜗牛酶 和0.6M MgCl2，经由0.22μm的无菌过滤器过滤除菌。将上述原生质体反应液通过无菌滤布进行过滤。5000rpm，4℃离心收集原生质体。用冰预冷的STC，洗涤一次，把原生质体重悬于预冷的STC中，并用STC将原生质体浓度调整为5×107个/mL，得到原生质体悬液。
向140μL上述原生质体悬液中加入10μL的UecdJ-hph-DecdJ片段，再加入50μL的PSTC，轻轻混匀，冰浴25min。加入1mL PSTC，混匀后室温放置20min；然后与10mL顶层琼脂混合后倾注于3块再生筛选培养基平板PDA-SH上，在30℃、黑暗条件下培养3-5天，得到转化子。
从转化筛选平板上挑取具有潮霉素抗性转化子转移至PDA-H上，在25℃培养4-6天。提取转化子基因组，利用外部引物UecdJ-F(5’-ctcggtctttatagtgcgtg-3’)和DecdJ-R(5’-atgaggatatgactgctctgg-3’)对转化子基因组进行PCR验证，能扩增出大小约为4.4kb的条带的为阳性转化子，而Coleophoma sp.MEFC009只能扩增出大小约为3.0kb的条带，图8结果表明获得了3个阳性转化子，说明在基因ecdJ位置发生了同源重组，整合了外源片段UecdJ-hph-DecdJ，将阳性菌株定义为ARTP-7-ΔecdJ。
将3株工程菌株ARTP-7-ΔecdJ和对照菌株ARTP-7接种于PDA固体平板上，25℃培养5-7天。取1～2mL无菌生理盐水于PDA平板中，用无菌小毛笔刷洗下孢子，将洗下的孢子用300～500目滤布过滤，收集孢子悬浮液并用孢子计数仪计数，加大(108CFU/mL)孢子悬浮液接种量于50mL的种子培养基，25℃，220rpm，摇床培养2天。将上述培养的种子液取5mL到50mL的发酵培养基，25℃，220rpm，发酵培养延长到13天，每个菌株设置3个平行，整个发酵过程中为避免蒸发需要控制摇床湿度。从每一瓶发酵液中取1mL，加入等体积的甲醇，漩涡震荡萃取1h，离心，取上清。用0.22μm的有机滤器过滤处理样品，并通过HPLC对处理后的样品进行分析。HPLC分析方法为：液相色谱柱为Agilent C-18反向柱883975-902(4.6×150mm，5μm)；流动相为A：0.05％(体积比)甲酸水溶液，流动相B：0.05％(体积比)甲酸乙腈溶液，流速为1mL/min，紫外检测波长：210nm，30℃，总洗脱时间为25min。梯度洗脱条件：0-5min，流动相B占流动相的体积由5％线性上升到40％，5-15min，流动相B占流动相的体积由40％线性上升到60％，15-20min，流动相B占流动相的体积由60％线性上升到100％。结果如图9所示；与出发菌株相比，ARTP-7-ΔecdJ中的棘白菌素B(ECB)完全消失，说明基因ecdJ与棘白菌素B的合成相关。
实施例8.过表达转录调控因子EcdJ工程菌株的构建
以A.nidulans ARTP-7的基因组为模板，利用引物ecdJ-F(5’-acaactcatcaatcatcacaATGCACTTCGCAGAGAGCGT-3’)和ecdJ-R(5’-caaaattcttcatttatttaCTATACTTGCTTACATAGGC-3’)进行PCR扩增，获得大小约为2.0kb大小的片段ecdJ。通过一步克隆酶连接到含有强启动子PgpdAt的线性化质粒PUC19上，将构建好的质粒导入大肠杆菌中增加拷贝数。以构建好的质粒为模板，利用引物PgpdAt-F(5’-ccgtagggatggccataatg-3’)和Tpgk-R(5’-attgcagcgcacaagtca-3’)进行PCR扩增，获得大小约为3.6kb大小的片段PgpdAt-ecdJ-TpgK。
以A.nidulans ARTP-7为出发菌，取1～2mL无菌生理盐水于PDA平板中，用无菌小毛笔刷洗下孢子，将洗下的孢子用300～500目滤布过滤，收集孢子悬浮液并用孢子计数仪计数，108CFU/mL孢子悬浮液接种量于50mL的种子培养基，25℃，220rpm，摇床培养1天，将培养基与菌丝体一起倒入50mL的无菌离心管中，4500rpm，离心收集菌丝。用0.6M MgCl2对菌丝体进行清洗2次。将菌丝体清洗至白色，称取1g菌丝，加入10-20mL酶解液，30℃，100-200rpm处理2-5h。酶解液成分为：0.6-1％溶壁酶、0.6-1％蜗牛酶和0.6M MgCl2，经由0.22μm的无菌过滤器过滤除菌。将上述原生质体反应液通过无菌300-500目滤布进行过滤。3000-5000rpm，4℃，离心20-40min收集原生质体。用冰预冷的STC，洗涤一次，把原生质体重悬于预冷的STC中，并用STC将原生质体浓度调整为5×107个/mL，得到原生质体悬液。
向100-200μL上述原生质体悬液中加入PgpdAt-ecdJ-Tpgk片段和hph片段，再加入50-100μL的PSTC，轻轻混匀，冰浴10-50min。加入0.5-2mL PSTC，混匀后室温放置10-40min；然后与10mL顶层琼脂混合后倾注于3块再生筛选培养基平板PDA-SH上，在30℃、黑暗条件下培养5-7天，得到转化子。
从转化筛选平板上挑取具有潮霉素抗性转化子转移至PDA-H上，在25℃培养5-7天进行传代培养，连续传代3次。选取10个转化子进行分离纯化，对纯化后的转化子基因组利用引物PgpdAt-F(5’-ccgtagggatggccataatg-3’)和Tpgk-R(5’-attgcagcgcacaagtca-3’)进行PCR验证，能扩增出大小约为3.6kb的条带为阳性转化子(图10)。
将过表达ecdJ的工程菌株和对照菌株A.nidulans ARTP-7接种于PDA固体平板上，25℃培养5-7天。取1～2mL无菌生理盐水于PDA平板中，用无菌小毛笔刷洗下孢子，将洗下的孢子用300～500目滤布过滤，收集孢子悬浮液并用孢子计数仪计数，加大(108CFU/mL)孢子悬浮液接种量于50mL的种子 培养基，25℃，220rpm，摇床培养2天。将上述培养的种子液取5mL到50mL的发酵培养基，25℃，220rpm，发酵培养延长到13天，每个菌株设置3个平行，整个发酵过程中为避免蒸发需要控制摇床湿度。从每一瓶发酵液中取1mL，加入等体积的甲醇，漩涡震荡萃取1h，离心，取上清。用0.22μm的有机滤器过滤处理样品，并通过HPLC对处理后的样品进行分析。
HPLC分析方法为：液相色谱柱为Agilent C-18反向柱883975-902(4.6×150mm，5μm)；流动相为A：0.05％(体积比)甲酸水溶液，流动相B：0.05％(体积比)甲酸乙腈溶液，流速为1mL/min，紫外检测波长：210nm，30℃，总洗脱时间为25min。梯度洗脱条件：0-5min，流动相B占流动相的体积由5％线性上升到40％，5-15min，流动相B占流动相的体积由40％线性上升到60％，15-20min，流动相B占流动相的体积由60％线性上升到100％。结果如图11所示，与野生型菌株ARTP-7相比，过表达ecdJ的工程菌株ARTP-7::ecdJ中化合物棘白菌素B的产量显著提高，与对照菌株ARTP-7相比提高了83.3％左右。
实施例9.在KB01菌株中过表达ctg12_1653提高纽莫康定B0的产量
以KB01(Glarea lozoyensis ATCC 74030)的基因组为模板，利用引物1653-F(5’-ACAACTCATCAATCATCACAATGGACCTTTTCCAAAGC-3’)和1653-R(5’-TCTTCATTTATTTATCTAGATCATACTGTTTCGCAGAG-3’)进行PCR扩增，获得大小约为2.2kb大小的ctg12_1653片段(本实施例方式中，ctg12_1653简称为1653)，ctg12_1653的核酸序列和氨基酸序列分别如SEQ ID No.5和SEQ ID No.6所示；通过一步克隆酶连接到含有强启动子PgpdAt的线性化质粒PUC19-ZX上，将构建好的质粒导入大肠杆菌中增加拷贝数。以构建好的质粒为模板，用引物PgpdAf(5’-CCGTAGGGATGGCCATAATG-3’)和hphr(5’-CAATTATCTTTGCGAACCCAGG-3’)进行PCR扩增，获的6kb的过表达片段PgpdAt-1653-Tpgk-hph。
将工程菌株KB01接种于PDA平板，28℃培养4～6天。用接种铲挖取1cm×3cm的菌块，加入1mL无菌水，用核酸提取仪破碎，接种到50mL的种子培养基，于250mL的三角瓶中，220rpm，25℃进行摇床培养。24～48h后离心收集菌丝，5000rpm，4℃，5min。用无菌单层100目尼龙布过滤收集长出的菌丝。用1M KCl对菌丝体进行清洗2次。压干后置于无菌的50mL三角瓶中，根据菌丝重量加入适量酶解液(每1g菌丝加入10mL酶解液)，在30℃、130rpm处理1～3h。酶解液成分为：0.5～1％溶壁酶、0.5～1％蜗牛酶和1M KCl，经由0.22μm的无菌过滤器过滤除菌。将上述酶解后的混合液用500目尼龙布或擦镜纸过滤，收集滤液。4000rpm，4℃离心收集原生质体。用冰预冷的STC，洗涤一次，把原生质体重悬于预冷的STC中，并用STC将原生质体浓度调整为5×107个/mL，得到原生质体悬液。
向200μL上述原生质体悬液中加入过表达片段PgpdAt-1653-Tpgk-hph，再加入50μL的PSTC，轻轻混匀，冰浴30min。加入1mL PSTC，混匀后室温放置20min；然后与10mL顶层琼脂混合后倾注于3块再生筛选培养基平板PDA-SH上，在30℃、黑暗条件下培养5-7天，得到转化子。
从转化筛选平板上挑取具有潮霉素抗性转化子转移至PDA-H上，在25℃培养5-7天进行传代培养，连续传代3次。选取12个转化子进行分离纯化，对纯化后的转化子基因组利用引物PgpdAt-F(5’-ccgtagggatggccataatg-3’)和Tpgk-R(5’-attgcagcgcacaagtca-3’)进行PCR验证，能扩增出大小约为3.6kb的条带为阳性转化子。
将过表达ctg12_1653的工程菌株和对照菌株KB01接种于PDA固体平板上，25℃培养5-7天。挑取少量的菌丝，利用核酸提取仪(-24)对菌丝进行破碎，将破碎后的菌丝接种于50mL KB01的种子培养基(250mL三角瓶)，25℃，220rpm，摇床培养96-120h。将上述培养的种子液取10mL到KB01的发酵培养基，25℃，220rpm，摇床培养11～12天，每个菌株设置3个平行。从每一瓶发酵液中取2mL，加入8mL的95％乙醇，超声萃取30min，离心，取上清。用0.22μm的有机滤器过滤处理样品，并通过HPLC对处理后的样品进行分析。
HPLC分析方法为：HPLC分析方法为：液相色谱柱为Agilent C-18反向柱883975-902(4.6×150mm，5μm)；流动相为A：水溶液，流动相B：乙腈溶液，流速为1mL/min，紫外检测波长：210nm，30℃，总洗脱时间为10min。洗脱条件：30～60％的流动相A。如图13所示，野生型KB01菌株中纽莫康定B0的产量为1623mg/L，工程菌株KB01::1653中化合物纽莫康定B0的产量最高，为2809mg/L，与对照菌株野生型KB01相比纽莫康定B0的产量提高了75％；图13中，KB01为野生型菌株，KB01::1653为过表达ctg12_1653的KB01菌株。
虽然本发明已以较佳的实施例公开如上，但其并非用以限定本发明，在不脱离本发明精神和范围内，本领域技术人员都可以在此基础上做出各种改动与变型，因此，本发明的保护范围应该以权利要求书所界定的为准。

Claims (10)

  1. 一种高产棘白菌素类化合物的基因工程菌株，所述基因工程菌株为在出发菌株中过表达转录因子而得到的基因工程菌株；所述转录因子的氨基酸序列与SEQ ID No.2或SEQ ID No.4或SEQ ID No.6相比具有至少70％的序列同一性。
  2. 根据权利要求1所述的基因工程菌株，其特征在于，
    所述棘白菌素类化合物为FR901379，所述转录因子的氨基酸序列与SEQ ID No.2相比具有至少70％的序列同一性，所述基因工程菌株的出发菌株为鞘茎点霉属真菌(Coleophoma sp.)；
    或者，
    所述棘白菌素类化合物为棘白菌素B，所述转录因子的氨基酸序列与SEQ ID No.4相比具有至少70％的序列同一性，所述基因工程菌株的出发菌株为Aspergillus nidulans、Aspergillus pachycristatu、Emericella nidulans、A.delacroxii、E.rugulosa或E.nidulans，所述出发菌株能够产生棘白菌素B；
    或者，
    所述棘白菌素类化合物为纽莫康定B0，所述转录因子的氨基酸序列与SEQ ID No.6相比具有至少70％的序列同一性，所述基因工程菌株的出发菌株为Glarea sp.。
  3. 一种制备高产棘白菌素类化合物的基因工程菌株的方法，所述方法包括在出发菌株中过表达权利要求1-2任一权利要求中的转录因子从而制备得到所述基因工程菌株的步骤。
  4. 根据权利要求3所述的方法，其特征在于，
    所述棘白菌素类化合物为FR901379，所述转录因子的氨基酸序列与SEQ ID No.2相比具有至少70％的序列同一性，所述基因工程菌株的出发菌株为鞘茎点霉属真菌；
    或者，
    所述棘白菌素类化合物为棘白菌素B，所述转录因子的氨基酸序列与SEQ ID No.4相比具有至少70％的序列同一性，所述基因工程菌株的出发菌株为Aspergillus nidulans、Aspergillus pachycristatu、Emericella nidulans、A.delacroxii、E.rugulosa或E.nidulans；
    或者，
    所述棘白菌素类化合物为纽莫康定B0，所述转录因子的氨基酸序列与SEQ ID No.6相比具有至少70％的序列同一性，所述基因工程菌株的出发菌株为Glarea sp.。
  5. 提高棘白菌素类化合物产量的转录因子或包含所述转录因子的生物材料在制备高产棘白菌素类化合物的基因工程菌株中的应用；其特征在于，
    所述转录因子的氨基酸序列与SEQ ID No.2或SEQ ID No.4或SEQ ID No.6相比具有至少70％的序列同一性；
    所述生物材料选自：所述转录因子的编码基因，或者包含所述编码基因的载体，或者包含所述载体的宿主细胞。
  6. 根据权利要求5所述的应用，其特征在于，
    所述棘白菌素类化合物为FR901379，所述转录因子的氨基酸序列与SEQ ID No.2相比具有至少70％的序列同一性；
    或者，
    所述棘白菌素类化合物为棘白菌素B，所述转录因子的氨基酸序列与SEQ ID No.4相比具有至少70％的序列同一性；
    或者，
    所述棘白菌素类化合物为纽莫康定B0，所述转录因子的氨基酸序列与SEQ ID No.6相比具有至少70％的序列同一性。
  7. 一种制备棘白菌素类化合物的方法，所述方法包括利用权利要求1-2任一所述的基因工程菌株进行发酵的步骤；任选的，所述方法还包括分离/纯化所述棘白菌素类化合物的步骤。
  8. 权利要求1-2任一所述的基因工程菌株，或权利要求3或4所述方法制备得到的基因工程菌株在生产棘白菌素类化合物中的应用；所述棘白菌素类化合物为FR901379、棘白菌素B或纽莫康定B0。
  9. 一种提高目的菌株中棘白菌素类化合物产量的方法，所述方法包括在目的菌株中过表达权利要求1-2任一权利要求中的转录因子的步骤。
  10. 根据权利要求9所述的方法，其特征在于，
    所述棘白菌素类化合物为FR901379，所述转录因子的氨基酸序列与SEQ ID No.2相比具有至少70％的序列同一性，所述目的菌株为鞘茎点霉属真菌；
    或者，
    所述棘白菌素类化合物为棘白菌素B，所述转录因子的氨基酸序列与SEQ ID No.4相比具有至少70％ 的序列同一性，所述目的菌株为Aspergillus nidulans、Aspergillus pachycristatu、Emericella nidulans、A.delacroxii、E.rugulosa或E.nidulans；
    或者，
    所述棘白菌素类化合物为纽莫康定B0，所述转录因子的氨基酸序列与SEQ ID No.6相比具有至少70％的序列同一性，所述目的菌株为Glarea sp.。
PCT/CN2023/106918 2022-07-18 2023-07-12 提高棘白菌素类化合物产量的转录因子及其应用 WO2024017105A1 (zh)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102443560A (zh) * 2010-10-13 2012-05-09 上海医药工业研究院 一种高效转化棘白菌素b的基因工程菌及其制备方法
CN103289901A (zh) * 2012-02-22 2013-09-11 上海来益生物药物研究开发中心有限责任公司 棘白菌素高产菌株及其应用
CN105648000A (zh) * 2014-11-19 2016-06-08 重庆乾泰生物医药有限公司 一种棘白菌素b微生物酶转化方法
CN105734098A (zh) * 2016-03-25 2016-07-06 浙江工业大学 一种提高棘白菌素b产量的方法
CN109593661A (zh) * 2017-09-30 2019-04-09 上海医药工业研究院 一种产棘白菌素b的基因工程菌及其构建方法和应用
CN109897843A (zh) * 2019-03-01 2019-06-18 浙江工业大学 一种重组棘白菌素b脱酰基酶突变体及应用
CN113215185A (zh) * 2021-04-22 2021-08-06 浙江工业大学 一种用于重组表达棘白菌素b脱酰基酶的重组基因序列

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102443560A (zh) * 2010-10-13 2012-05-09 上海医药工业研究院 一种高效转化棘白菌素b的基因工程菌及其制备方法
CN103289901A (zh) * 2012-02-22 2013-09-11 上海来益生物药物研究开发中心有限责任公司 棘白菌素高产菌株及其应用
CN105648000A (zh) * 2014-11-19 2016-06-08 重庆乾泰生物医药有限公司 一种棘白菌素b微生物酶转化方法
CN105734098A (zh) * 2016-03-25 2016-07-06 浙江工业大学 一种提高棘白菌素b产量的方法
CN109593661A (zh) * 2017-09-30 2019-04-09 上海医药工业研究院 一种产棘白菌素b的基因工程菌及其构建方法和应用
CN109897843A (zh) * 2019-03-01 2019-06-18 浙江工业大学 一种重组棘白菌素b脱酰基酶突变体及应用
CN113215185A (zh) * 2021-04-22 2021-08-06 浙江工业大学 一种用于重组表达棘白菌素b脱酰基酶的重组基因序列

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