WO2024017105A1 - Transcription factor for improving yield of echinocandin compounds and use thereof - Google Patents

Abstract

The present invention provides a transcription factor for improving the yield of echinocandin compounds. The overexpression of a transcription factor in a starting strain can improve the yield of echinocandin compounds. Further provided is a use of said transcription factor in the preparation of a genetically engineered strain having high echinocandin compound yield; and further provided is a use of the genetically engineered strain in the preparation of echinocandin compounds.

Description

Transcription factors that increase the production of echinocandins and their applications Technical field

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.

Background technique

Echinocandins prodrugs are a type of cyclic lipopeptide compounds produced by filamentous fungi. These drugs can selectively inhibit the activity of β-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.

At present, the fermentation production process of echinocandin drug precursors is faced with problems such as low concentration of main products and many by-products, which makes the production cost of this type of drugs remain high. Even though this type of drugs has good antifungal effects, However, due to its high production cost and expensive price, it is generally not used.

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. Although the 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. This study discovered for the first time a specific transcriptional regulatory factor in the biosynthetic gene cluster of micafungin precursor FR901379, and through two strategies: replacing the transcription factor promoter and increasing the copy number, the production of FR901379 was significantly improved. This study provides An effective strategy to effectively increase the production of echinocandins.

Contents of the invention

On the one hand, 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.

In one embodiment, the transcription factor is the transcription factor McfJ, and 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, preferably, the McfJ source For fungi of the genus Coleophoma, for example, Coleophoma sp. MEFC009. Preferably, the amino acid sequence of McfJ is shown in SEQ ID No. 2.

In one embodiment, the transcription factor is the transcription factor EcdJ, and 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, preferably, the EcdJ source For Aspergillus nidulans, for example, Aspergillus nidulans ARTP-7 with deposit number CGMCC No. 40073. Preferably, 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.

In one embodiment, 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. Preferably, the amino acid sequence of ctg12_1653 is shown in SEQ ID No. 6.

On the other hand, 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.

On the other hand, the present invention also provides genes encoding the above-mentioned transcription factors.

On the other hand, the present invention also provides a vector containing the above gene, or a host cell containing the vector.

In one embodiment, 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, pUC series vectors (eg, pUC18, pUC19), pQE-30, pXH2-1, pXH-43, pTRII, pGSF957.

In one embodiment, the host cell is selected from the group consisting of E. coli DH5α, 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.

On the other hand, 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. In one embodiment, the echinocandin compound is FR901379, echinocandin B or niumocandin B ₀ .

In one embodiment, 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.

In one embodiment, 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.

In one embodiment, 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 .

On the other hand, 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 In the method, the transcription factor is McfJ, the echinocandins are FR901379, and the starting strain of the genetic engineering strain is a fungus of the genus Coleophoma, preferably Coleophoma sp. MEFC009; In other embodiments, the transcription factor is EcdJ, the echinocandin compound is echinocandin B, and 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; in other embodiments, the transcription factor is ctg12_1653, and 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.

On the other hand, 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. In one embodiment, the transcription factor is McfJ, the echinocandins are FR901379, and the starting strain of the genetically engineered strain is a fungus of the genus Coleophoma, preferably Coleophoma sp. ) MEFC009; In other embodiments, the transcription factor is EcdJ, the echinocandin compound is echinocandin B, and 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 other embodiments, the transcription factor is ctg12_1653, the echinocandin compound is niumocandin B ₀ , and the genetic engineering The starting strain of the strain is Glarea sp., preferably, Glarea lozoyensis.

On the other hand, 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. In one embodiment, the transcription factor is McfJ, the echinocandins are FR901379, and the starting strain of the genetically engineered strain is a fungus of the genus Coleophoma, preferably Coleophoma sp. ) MEFC009; In other embodiments, the transcription factor is EcdJ, the echinocandin compound is echinocandin B, and 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; in other embodiments, the transcription factor is ctg12_1653, the echinocandin compound is niumocandin B ₀ , and 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. In one embodiment, 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.

In one embodiment, 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.

In another embodiment, the overexpression includes the step of introducing additional copies of the transcription factor into the starting strain.

In one embodiment, 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.

In another embodiment, 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, In the embodiment, 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.

In a preferred embodiment, 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 .

In other embodiments, 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 literature Kim JG et al.Genetic transformation of Monascus purpureus DSM1379.Biotechnol Lett, 2003, 25:1509–1514 disclosed Pmgpd and Ptrp (ie PtrpC). The literature 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 disclosed PgpdA. Document 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 disclosed Ppgk.

On the other hand, 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, and the starting strain of the genetically engineered strain is a fungus of the genus Coleophoma sp., preferably Coleophoma sp. MEFC009; in other embodiments, the transcription factor is EcdJ, and the echinacea The cannabinoid compound is echinocandin B, and 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 In an embodiment, the transcription factor is ctg12_1653, the echinocandin compound is neomocontin B ₀ , and the starting strain of the genetically engineered strain is Glarea sp., preferably, Glarea lozoyensis.

On the other hand, 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.

In one embodiment, 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, and the genetically engineered strain The starting strain of the strain is a fungus of the genus Coleophoma, preferably Coleophoma sp. MEFC009.

In other embodiments, 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.

In other embodiments, 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.

On the other hand, 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. In one embodiment, the echinocandins are FR901379, the transcription factor is McfJ, and the target strain is a fungus of the genus Coleophoma, preferably Coleophoma sp. MEFC009. In other embodiments, the echinocandin compound is echinocandin B, the transcription factor is EcdJ, and the target strain is Aspergillus nidulans, Aspergillus pachycristatu, Emericella nidulans, A.delacroxii, E.rugulosa or E. nidulans, preferably Aspergillus nidulans. In other embodiments, the echinocandins are niumocandin B ₀ , the transcription factor is ctg12_1653, and the target strain is Glarea sp., preferably, Glarea lozoyensi.

Description of drawings

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.

Figure 2 shows the HPLC analysis results of the fermentation product of the gene mcfJ deletion strain; MEFC009: MEFC--ΔmcfJ is the mcfJ deletion strain; the control strain Coleophoma sp. MEFC009; 1: FR901379.

Figure 3 shows the transcript level analysis of FR901379 biosynthetic genes; MEFC-ΔmcfJ is the gene mcfJ deletion strain, MEFC009 is the control strain Coleophoma sp.MEFC009, 8763_g: actin encoding gene, and 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-Δku80, 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. 1, 2: control strain Coleophoma sp. MEFC009; 3, 4: engineered strain MEFC::mcfJ ;8763_g: Actin-encoding gene.

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.

Figure 9 HPLC analysis results of the fermentation product of A.nidulans-ΔecdJ gene ecdJ deletion strain; ARTP-7-ΔecdJ is the gene ecdJ deletion strain, and ARTP-7 is the control strain.

Figure 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.

Figure 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.

Figure 12 PCR verification of the transformant genome obtained by overexpressing the transcriptional regulatory factor ctg12_1653; 1-4: Transformant genome.

Figure 13 Analysis of the B ₀ production of niumocontin in the fermentation broth of the engineering strain overexpressing the transcriptional regulator ctg12_1653 and the control strain KB01.

Detailed ways

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

The nucleic acid sequences and amino acid sequences of McfJ, EcdJ and ctg12_1653 involved in this embodiment are shown in the following table:

In the present invention, 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 ). (Trace 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.

STC: 1M sorbitol, 50mM Tris-HCl (pH 8.0), 50mM CaCl ₂ .

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.

In this embodiment, 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-ΔmcfJ with knockout gene mcfJ

In the FR901379 biosynthetic gene cluster, there is the gene 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.

Using the genome of wild-type Coleophoma sp.MEFC009 as a template, 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 1.4kb can be amplified. 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 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.

Using Coleophoma sp.MEFC009 as the starting strain, first take a small amount of mycelium from the PDA plate, crush it with a hand-held homogenizer, take 1mL of seed liquid and inoculate it into 50mL of seed culture medium, and inoculate it in a 250mL Erlenmeyer flask at 220rpm and 25℃. Shaker culture. After 2 days, the hyphae were collected by centrifugation. 5000rpm, 4℃, 5min. Break the mycelium again with a homogenizer, take 0.5mL-2mL seed liquid and inoculate it into 50mL seed culture medium. Cultivate it for 1 day under the same conditions. Pour the culture medium and mycelium into a 50mL sterile centrifuge tube. Medium, 5000rpm, centrifuge to collect mycelium. Wash the mycelium twice with 0.6M _MgSO4 . Wash the mycelium until white, weigh 1g of mycelium, add 10 mL of enzymatic hydrolyzate, and process at 30°C and 100 rpm for 1-4 hours. 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 μm 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.

Add 10 μL of UmcfJ-hph-DmcfJ 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 place at room temperature for 20 minutes; then mix with 10 mL of top agar and pour onto three regeneration screening medium plates, PDA-SH, and culture at 30°C in the dark for 5-7 days to obtain transformants.

Select hygromycin-resistant transformants from the transformation screening plate and transfer them to PDA-H. Culture them at 25°C for 5-7 days for subculture, and continue to subculture them for 3 generations. Three stably passaged transformants 2#, 5#, and 8# were selected for single spore isolation and purification, and the genomes of the transformants after single spore isolation were extracted. Use external primers UmcfJ-F (5'-ccggtggcttgaaagatttc-3') and DmcfJ-R (5'-aggaggactcgaaatcaaag-3') to perform PCR verification on the transformant genome. 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-ΔmcfJ.

Example 2. Fermentation verification of engineering strain MEFC-ΔmcfJ

Three engineering strains Coleophoma sp.-ΔmcfJ-2#, 5#, 8# and the control strain Coleophoma sp.MEFC009 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, and inoculate the broken mycelium into 50 mL Coleophoma sp. seed culture medium (250 mL Erlenmeyer flask), 25°C, 220 rpm, and culture on a shaker for 45-48 hours. Take 5 mL to 50 mL of Coleophoma sp. fermentation medium (250 mL Erlenmeyer flask) from the above-mentioned cultured seed liquid, and culture it on a shaking table at 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 μm 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, 5μm); 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. Gradient elution conditions: 0-5min, the volume of mobile phase B in the mobile phase increases linearly from 5% to 24%, 5-35min, the volume of mobile phase B in the mobile phase increases linearly from 24% to 62%, 35-37min , the volume of mobile phase B increased linearly from 62% to 100%. The results are shown in Figure 2; compared with the starting strain, FR901379 (compound 1) and its intermediates in MEFC-ΔmcfJ completely disappeared, indicating that the gene mcfJ is related to the synthesis of FR901379.

Since 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.

Example 3. Analysis of transcription levels of FR901379 biosynthesis-related genes

3.1 Extraction of RNA

Based on the above results, it is speculated that mcfJ is likely to be a transcriptional regulator. To test this hypothesis, RNA was extracted from wild-type Coleophoma sp.MEFC009 and mcfJ deletion strains. 1) Place the fresh bacterial cells in a mortar, add liquid nitrogen and grind until they are ground into powder. Transfer 50-100 mg of bacterial cells to a sterilized 1.5 mL EP tube, add 450 μL Buffer RL (add 50×DTT in advance), and pipette repeatedly with a pipette until there is no obvious precipitation. 2) Centrifuge the lysate at 12,000 rpm and 4°C for 5 minutes. 3) Carefully pipet the supernatant into a new 1.5mL sterilized EP tube. 4) Add 1/2 volume of absolute ethanol to the supernatant or mixed solution in the sample lysis step, and use a pipette to mix the solution evenly. 5) Immediately transfer the entire mixture (including precipitation) to the RNA spin column. 6) Centrifuge at 12,000 rpm for 1 minute and discard the filtrate. Place the RNA spin column back into the 2 mL collection tube. 7) Add 500 μL of Buffer RWA to the RNA spin column, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate. 8) Add 600 μL of Buffer RWB to the RNA spin column, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate. 9) Add 50μL DNase I reaction solution to the center of the RNA spin column membrane and let it stand at room temperature for 15 minutes. 10) Add 600 μL of Buffer RWB to the RNA spin column, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate. 11) Replace the RNA spin column on the 2 mL collection tube and centrifuge at 12,000 rpm for 2 minutes. 12) Place the RNA spin column on a 1.5 mL RNase-free collection tube, add 50 μL 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.

3.2 Reverse transcribe RNA into cDNA

(1) First digest the remaining DNA from the RNA extracted in 3.1. The reaction system is as follows: 2.0μL of 5×gDNA Eraser buffer, 1.0μL of gDNA Eraser, 1.0μg of total RNA. Use RNase-free water to make up the reaction system to 10 μL, 42°C, place for 5 minutes.

(2) Reverse transcription: Add 4.0μL of 5×PrimerScript II buffer, 0.5μL of RNase inhibitor, 1.0μL of reverse transcriptase, 1μL of RT Primer Mix to the reaction solution in (1), and use RNase-free Make up the reaction system to 20 μL with water, place at 37°C for 15 minutes, and then at 85°C for 5 seconds.

3.3 Analysis of transcription levels of genes related to FR901379 synthesis

Design primers in the coding region of the gene responsible for FR901379 synthesis: 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') and mcfACDS-F(5'-ttcgaacctcggagaatcgag-3')/mcfACDS-R (5'-gctggtcacttccagactaca'). 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.

Example 4. Construction of recombinant strain with promoter replacement of transcription regulator McfJ

4.1 Construction of transcriptional regulator McfJ promoter replacement expression cassette

Using the genome of Coleophoma sp.MEFC009 as a template, 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. 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 hygromycin of approximately 2.2 kb. 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. 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.

4.2 Construction of recombinant strain with replacement of transcription regulator McfJ promoter

Using Coleophoma sp.MEFC009-Δku80 as the starting strain, first take a small amount of mycelium from the PDA plate, crush it with a hand-held homogenizer, take 1mL of seed liquid and inoculate it into 50mL of seed culture medium, place it in a 250mL Erlenmeyer flask, 220rpm, 25 ℃ for shaking culture. After 2 days, the hyphae were collected by centrifugation. 5000rpm, 4℃, 5min. Break the mycelium again with a homogenizer, take 0.5mL-2mL seed liquid and inoculate it into 50mL seed culture medium. Cultivate it for 1 day under the same conditions. Pour the culture medium and mycelium into a 50mL sterile centrifuge tube. Medium, 5000rpm, centrifuge to collect mycelium. Wash the mycelium twice with 0.6M _MgSO4 . Wash the mycelium until white, weigh 1g of mycelium, add 10 mL of enzymatic hydrolyzate, and process at 30°C and 100 rpm for 1-4 hours. 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 μm 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.

Add UMcfJ-2-hph-PgpdAt-DMcfJ-2 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 place at room temperature for 20 minutes; then mix with 10 mL of top agar and pour onto three regeneration screening medium plates, PDA-SH, and culture at 30°C in the dark for 5-7 days to obtain transformants.

Select hygromycin-resistant transformants from the transformation screening plate and transfer them to PDA-H, culture them at 25°C for 5-7 days, and subculture them three times. Eight transformants were selected for isolation and purification. The purified transformant genome was verified by PCR using primers UMcfJ-2F (5'-tacaagcactaatgtaatcg-3') and DMcfJ-2R (5'-ACATGCCGTGCTGGCGACAT-3'). It can be amplified. 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.

Example 5. Construction of recombinant strains with increased copy number of the transcription regulator McfJ

Using the genome of Coleophoma sp.MEFC009 as a template, 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.

Using Coleophoma sp.MEFC009 as the starting strain, first take a small amount of mycelium from the PDA plate, crush it with a hand-held homogenizer, take 1mL of seed liquid and inoculate it into 50mL of seed culture medium, and inoculate it in a 250mL Erlenmeyer flask at 220rpm and 25℃. Shaker culture. After 2 days, the hyphae were collected by centrifugation. 5000rpm, 4℃, 5min. Break the mycelium again with a homogenizer, take 0.5mL-2mL seed liquid and inoculate it into 50mL seed culture medium. Cultivate it for 1 day under the same conditions. Pour the culture medium and mycelium into a 50mL sterile centrifuge tube. Medium, 5000rpm, centrifuge to collect mycelium. Wash the mycelium twice with 0.6M _MgSO4 . Wash the mycelium until white, weigh 1g of mycelium, add 10 mL of enzymatic hydrolyzate, and process at 30°C and 100 rpm for 1-4 hours. 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 μm 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.

Add P-McfJ-T fragment and hph fragment to 200 μL of the above protoplast suspension, then add 50 μL of PSTC, mix gently, and incubate on ice for 30 min. Add 1mL PSTC, mix well and place at room temperature for 20 minutes; then mix with 10mL top agar and pour into 3 blocks to regenerate On the screening medium plate PDA-SH, culture at 30°C and dark conditions for 5-7 days to obtain transformants.

Select hygromycin-resistant transformants from the transformation screening plate and transfer them to PDA-H, culture them at 25°C for 5-7 days, and subculture them three times. Transformants were selected and cultured in fermentation medium for 1.5 days, RNA was extracted, and its RNA was reverse transcribed into cDNA, using wild-type Coleophoma sp.MEFC009 as a control. The cDNAs of wild-type Acanthus sp. 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. When the cDNA of the engineered strain MEFC::mcfJ is used as the template, the concentration of the amplified band is much higher than that of the control group, indicating that the copy number of the gene mcfJ in the engineered strain MEFC::mcfJ is higher than that of the control. Strain Coleophoma sp.MEFC009.

Example 6. Fermentation verification of recombinant strain overexpressing the transcription regulator McfJ

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. Take 5 mL of the above cultured seed liquid into the fermentation medium of Coleophoma sp., and culture it on a shaker for 8 days at 25°C, 220 rpm, and set up 3 parallels for each strain. 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 μm 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, 5μm); 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. Gradient elution conditions: 0-5min, the volume of mobile phase B in the mobile phase increases linearly from 5% to 40%, 5-20min, the volume of mobile phase B in the mobile phase increases linearly from 40% to 62%, 20-25min , the volume of mobile phase B increased linearly from 62% to 100%. The HPLC analysis results are shown in Figure 6, from which it can be seen that compared with wild-type Coleophoma sp.MEFC009, the production of compound FR901379 in the engineered strains MEFC-PgpdAt-mcfJ and MEFC::mcfJ was significantly improved. The production of 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.

We have also found 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.

We overexpressed EcdJ and ctg12_1653 in A.nidulans ARTP-7 (strain deposit number: CGMCC No. 40073) and KB01 respectively, which can also increase the production of echinocandin B and nimocandin B _0. Specific implementation methods as follows:

Example 7. Construction of engineering strain ARTP-7-ΔecdJ with knockout gene 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.

Using the genome of A.nidulans ARTP-7 as a template, pfu DNA polymerase (Fermentas, Catalog No.: EP0501) was used for PCR amplification, using primers UecdJ-F (5'-ctcggtctttatagtgcgtg-3') and UecdJ-R ( 5'-tttacgcttgcgatcccgaaGAGGTCAAAGTTTAGAAAAT-3') can amplify the upstream sequence U-ecdJ of ecdJ with a size of about 1.2kb, using primers DecdJ-F (5'-tgggttcgcaaagataattgGGGCAAGCAGTCCATCGCCC-3') and DecdJ-R (5'-atgaggatatgactgctctgg- 3') The downstream sequence D-mcfJ of ecdJ with a size of 1.2kb can be amplified. 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 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.

Using 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. Wash the mycelium twice with 0.6M _MgCl2 . Wash the mycelium until white, weigh 1g of mycelium, add 15mL of enzymatic hydrolyzate, and process at 30°C and 130rpm for 2-3 hours. The components of the enzymatic solution are: 1% wall-lying enzyme, 0.6% helicase and 0.6M MgCl ₂ , filtered through a 0.22 μm sterile filter. Filter the above protoplast reaction solution through sterile 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.

Add 10 μL of UecdJ-hph-DecdJ fragment to 140 μL of the above protoplast suspension, then add 50 μL of PSTC, mix gently, and incubate on ice for 25 min. Add 1 mL of PSTC, mix well and leave at room temperature for 20 minutes; then mix with 10 mL of top agar and pour onto 3 regeneration screening medium plates PDA-SH, and culture at 30°C in the dark for 3-5 days to obtain transformants.

Select hygromycin-resistant transformants from the transformation screening plate, transfer them to PDA-H, and culture them at 25°C for 4-6 days. Extract the transformant genome and use external primers UecdJ-F (5'-ctcggtctttatagtgcgtg-3') and DecdJ-R (5'-atgaggatatgactgctctgg-3') to perform PCR verification on the transformant genome. The amplified size is about 4.4kb. The band is a positive transformant, while Coleophoma sp.MEFC009 can only amplify a band of about 3.0kb in size. The results in Figure 8 show that 3 positive transformants were obtained, indicating that homologous recombination occurred at the ecdJ position of the gene. , the exogenous fragment UecdJ-hph-DecdJ was integrated, and the positive strain was defined as ARTP-7-ΔecdJ.

The three engineering strains ARTP-7-ΔecdJ 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. Take 5 mL to 50 mL of fermentation medium from the above-mentioned cultured seed liquid, 25°C, 220 rpm, and extend the fermentation culture to 13 days. Set up 3 parallels for each strain. During the entire fermentation process, the shaker humidity needs to be controlled to avoid evaporation. Take 1 mL from each bottle of fermentation broth, add an equal volume of methanol, vortex and extract for 1 hour, centrifuge, and take the supernatant. The samples were filtered through a 0.22 μm 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, 5μm); 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℃, total elution time is 25min. Gradient elution conditions: 0-5min, the volume of mobile phase B in the mobile phase increases linearly from 5% to 40%, 5-15min, the volume of mobile phase B in the mobile phase increases linearly from 40% to 60%, 15-20min , the volume of mobile phase B occupied by mobile phase increases linearly from 60% to 100%. The results are shown in Figure 9; compared with the starting strain, echinocandin B (ECB) in ARTP-7-ΔecdJ completely disappeared, indicating that the gene ecdJ is related to the synthesis of echinocandin B.

Example 8. Construction of engineering strains overexpressing the transcription regulator EcdJ

Using the genome of A.nidulans ARTP-7 as a template, 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. Through one-step cloning enzyme, it is connected to the linearized plasmid PUC19 containing the strong promoter PgpdAt, and the constructed plasmid is introduced into E. coli to increase the copy number. Using the constructed plasmid as a template, 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.

Using 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. Wash the mycelium twice with 0.6M _MgCl2 . Wash the mycelium until white, weigh 1g of mycelium, add 10-20mL of enzymatic hydrolyzate, and process at 30°C and 100-200rpm for 2-5 hours. The components of the enzymatic hydrolyzate are: 0.6-1% wall-lysing enzyme, 0.6-1% helicase and 0.6M MgCl ₂ . Filter and sterilize through a 0.22 μm sterile filter. Filter the above protoplast reaction solution through sterile 300-500 mesh filter cloth. Centrifuge at 3000-5000 rpm, 4°C for 20-40 minutes to collect protoplasts. 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.

Add PgpdAt-ecdJ-Tpgk fragment and hph fragment to 100-200 μL of the above protoplast suspension, then add 50-100 μL of PSTC, mix gently, and incubate on ice for 10-50 min. Add 0.5-2mL PSTC, mix and place at room temperature for 10-40min; then mix with 10mL top agar and pour onto 3 regeneration screening medium plates PDA-SH, and culture at 30°C in the dark for 5-7 days to obtain Turn.

Select hygromycin-resistant transformants from the transformation screening plate and transfer them to PDA-H, culture them at 25°C for 5-7 days, and subculture them three times. Select 10 transformants for isolation and purification, and use primers PgpdAt-F (5'-ccgtagggatggccataatg-3') and Tpgk-R (5'-attgcagcgcacaagtca-3') to perform PCR verification on the purified transformant genome. It can be amplified. The band with a size of about 3.6 kb was a positive transformant (Figure 10).

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℃, 220rpm, shaker culture for 2 days. Take 5 mL to 50 mL of fermentation medium from the above-mentioned cultured seed liquid, 25°C, 220 rpm, and extend the fermentation culture to 13 days. Set up 3 parallels for each strain. During the entire fermentation process, the shaker humidity needs to be controlled to avoid evaporation. Take 1 mL from each bottle of fermentation broth, add an equal volume of methanol, vortex and extract for 1 hour, centrifuge, and take the supernatant. The samples were filtered through a 0.22 μm 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, 5μm); 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℃, total elution time is 25min. Gradient elution conditions: 0-5min, the volume of mobile phase B in the mobile phase increases linearly from 5% to 40%, 5-15min, the volume of mobile phase B in the mobile phase increases linearly from 40% to 60%, 15-20min , the volume of mobile phase B occupied by mobile phase increases linearly from 60% to 100%. The results are shown in Figure 11. Compared with the wild-type strain ARTP-7, the production of the compound echinocandin B in the engineered strain ARTP-7::ecdJ overexpressing ecdJ was significantly increased compared with the control strain ARTP-7. About 83.3%.

Example 9. Overexpression of ctg12_1653 in KB01 strain improves the production of neumocontin B ₀

Using the genome of KB01 (Glarea lozoyensis ATCC 74030) as a template, PCR amplification was performed using primers 1653-F (5'-ACAACTCATCAATCATCACAATGGACCTTTTCCAAAGC-3') and 1653-R (5'-TCTTCATTTATTTATCTAGATCATACTGTTTCGCAGAG-3'), and the obtained size was approximately 2.2 Kb-sized ctg12_1653 fragment (in this embodiment, 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. 6 respectively; it is connected to a strong promoter-containing protein through one-step cloning enzyme On the linearized plasmid PUC19-ZX of PgpdAt, the constructed plasmid was introduced into E. coli to increase the copy number. Using the constructed plasmid as a template, 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. Use an inoculation shovel to dig out a 1cm × 3cm bacterial mass, add 1mL of sterile water, crush it with a nucleic acid extractor, inoculate it into 50mL of seed culture medium, and culture it on a shaker at 220rpm and 25°C in a 250mL Erlenmeyer flask. After 24 to 48 hours, the mycelium was collected by centrifugation, 5000 rpm, 4°C, 5 min. Use a sterile single-layer 100-mesh nylon cloth to filter and collect the growing hyphae. 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 μm 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.

Add the overexpressed fragment PgpdAt-1653-Tpgk-hph to 200 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 place at room temperature for 20 minutes; then mix with 10 mL of top agar and pour onto three regeneration screening medium plates, PDA-SH, and culture at 30°C in the dark for 5-7 days to obtain transformants.

Select hygromycin-resistant transformants from the transformation screening plate and transfer them to PDA-H, culture them at 25°C for 5-7 days, and subculture them three times. 12 transformants were selected for isolation and purification. The purified transformant genome was verified by PCR using primers PgpdAt-F (5'-ccgtagggatggccataatg-3') and Tpgk-R (5'-attgcagcgcacaagtca-3'). It can be amplified. The band with a size of about 3.6kb is a positive transformant.

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. Take 10 mL of the above cultured seed liquid into the KB01 fermentation medium, culture it on a shaker at 25°C, 220 rpm for 11 to 12 days, and set up 3 parallels for each strain. Take 2 mL from each bottle of fermentation broth, add 8 mL of 95% ethanol, perform ultrasonic extraction for 30 min, centrifuge, and take the supernatant. The samples were filtered through a 0.22 μm organic filter and analyzed by HPLC.

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, 5μm); the mobile phase is A: aqueous solution, mobile phase B: acetonitrile solution, the flow rate is 1mL /min, UV detection wavelength: 210nm, 30℃, total elution time is 10min. Elution conditions: 30 to 60% mobile phase A. As shown in Figure 13, 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.

Although the present invention has been disclosed above in terms of preferred embodiments, they are not intended to limit the present invention. Those skilled in the art can make various changes and modifications on this basis without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims (10)

1. A genetically engineered strain with high production of echinocandin compounds, the genetically engineered strain is a genetically engineered strain obtained by overexpressing a transcription factor in the starting strain; the amino acid sequence of the transcription factor is consistent with SEQ ID No. 2 or SEQ ID No.4 or SEQ ID No.6 has at least 70% sequence identity.
2. The genetically engineered strain according to claim 1, characterized in that,

   The echinocandins are FR901379, the amino acid sequence of the transcription factor has at least 70% sequence identity compared with SEQ ID No. 2, and the starting strain of the genetically engineered strain is a fungus of the genus Coleoplasty (Coleophoma sp.);

   or,

   The echinocandin compound is echinocandin B, the amino acid sequence of the transcription factor has at least 70% sequence identity compared with SEQ ID No. 4, and 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 can produce echinocandin B;

   or,

   The echinocandins are neomocontin B ₀ , the amino acid sequence of the transcription factor has at least 70% sequence identity compared with SEQ ID No. 6, and the starting strain of the genetically engineered strain is Glarea sp. ..
3. A method for preparing a genetically engineered strain with high yield of echinocandin compounds, the method includes the step of overexpressing the transcription factor in any one of claims 1-2 in the starting strain to prepare the genetically engineered strain. .
4. The method according to claim 3, characterized in that:

   The echinocandins are FR901379, the amino acid sequence of the transcription factor has at least 70% sequence identity compared with SEQ ID No. 2, and the starting strain of the genetically engineered strain is a fungus of the genus Coleoplasty ;

   or,

   The echinocandin compound is echinocandin B, the amino acid sequence of the transcription factor has at least 70% sequence identity compared with SEQ ID No. 4, and the starting strain of the genetically engineered strain is Aspergillus nidulans , Aspergillus pachycristatu, Emericella nidulans, A.delacroxii, E.rugulosa or E.nidulans;

   or,

   The echinocandins are neomocontin B ₀ , the amino acid sequence of the transcription factor has at least 70% sequence identity compared with SEQ ID No. 6, and the starting strain of the genetically engineered strain is Glarea sp. ..
5. The application of a transcription factor that improves the production of echinocandins or a biological material containing the transcription factor in the preparation of a genetically engineered strain with high yield of echinocandins; characterized in that,

   The amino acid sequence of the transcription factor has at least 70% sequence identity compared with SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6;

   The biological material is selected from: a gene encoding the transcription factor, a vector containing the encoding gene, or a host cell containing the vector.
6. The application according to claim 5, characterized in that:

   The echinocandins are FR901379, and the amino acid sequence of the transcription factor has at least 70% sequence identity compared with SEQ ID No. 2;

   or,

   The echinocandin compound is echinocandin B, and the amino acid sequence of the transcription factor has at least 70% sequence identity compared with SEQ ID No. 4;

   or,

   The echinocandins are niumocandin B ₀ , and the amino acid sequence of the transcription factor has at least 70% sequence identity compared with SEQ ID No. 6.
7. A method for preparing echinocandins, which method includes the step of fermenting the genetically engineered strain described in any one of claims 1-2; optionally, the method further includes isolating/purifying the echinocandins. Procedure for cannabinoids.
8. The application of the genetically engineered strain of any one of claims 1-2, or the genetically engineered strain prepared by the method of claim 3 or 4 in the production of echinocandins; the echinocandins are FR901379, echinocandin B or niumocandin B ₀ .
9. A method for increasing the production of echinocandin compounds in a target strain, which method includes the step of overexpressing the transcription factor of any one of claims 1-2 in the target strain.
10. The method according to claim 9, characterized in that:

    The echinocandins are FR901379, the amino acid sequence of the transcription factor has at least 70% sequence identity compared with SEQ ID No. 2, and the target strain is a fungus of the genus Coleomyces;

    or,

    The echinocandins are echinocandins B, and the amino acid sequence of the transcription factor has at least 70% of the amino acid sequence of SEQ ID No. 4. Sequence identity, the target strain is Aspergillus nidulans, Aspergillus pachycristatu, Emericella nidulans, A.delacroxii, E.rugulosa or E.nidulans;

    or,

    The echinocandin compound is neumocontin B ₀ , the amino acid sequence of the transcription factor has at least 70% sequence identity compared with SEQ ID No. 6, and the target strain is Glarea sp.