DNA binding site of a fungal transcription factor Anij and application thereof

Abstract

The application belongs to the field of molecular biology technology, and particularly relates to a DNA binding site of a fungal transcription factor AniJ and application thereof. The application takes the echinocandin B gene cluster regulatory factor AniJ as a research object, uses a ptH-SpRY-ACBE base editor and an sgRNA library covering an aniA promoter, and combines 5-FOA screening to mine the AniJ binding motif RNGCTGAS in the core promoter region. By constructing an engineering strain containing a modified promoter (increasing the number of motifs), it is confirmed that the motif can significantly enhance the expression of aniA and the yield of echinocandin B. The application provides a new type of molecular tool for fungal natural product metabolic engineering.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to the DNA binding site of a fungal transcription factor AniJ and its application. Background Technology

[0002] Echinocandin B (ECB) is a six-membered cyclic peptide compound and an important precursor for the chemically modified synthesis of the clinical antifungal drug anidulafungin. These drugs exert broad-spectrum antifungal activity by inhibiting the synthesis of β-(1,3)-glucan, a major component of fungal cell walls, and are highly effective against invasive fungal infections such as drug-resistant Candida and Aspergillus. ECB has a complex structure and is currently only produced through fermentation by filamentous fungi. Its biosynthetic gene cluster contains genes encoding large non-ribosomal peptide synthases (NRPS), responsible for assembling the six-membered cyclic peptide backbone; in addition, the gene cluster also contains some oxygenases responsible for the hydroxylation and oxygenation of specific amino acid residues in the six-membered cyclic peptide.

[0003] exist Anidulans nidulans middle, ani The gene cluster is a known gene cluster responsible for ECB biosynthesis. Among them, AniA is the NRPS responsible for the assembly of the ECB six-membered cyclic peptide backbone and is also the rate-limiting enzyme in ECB biosynthesis; while AniJ is the regulatory enzyme. ani Specific transcription activators expressed by each gene in a gene cluster. However, AniJ transcription factors... ani The activation properties of genes within the gene cluster vary considerably, and overexpression of this transcription factor alone cannot achieve optimal ECB production. Currently... ani The promoters in gene clusters and their binding sites with the transcription factor AniJ are currently unclear, making it difficult to carry out ECB metabolic pathway modification based on transcriptional regulation optimization. There are no reports on how to identify the binding sites of the AniJ transcription factor in genes and apply them to promoter engineering to promote product enhancement. Summary of the Invention

[0004] To address the aforementioned problems, the purpose of this invention is to provide a DNA binding site for the fungal transcription factor AniJ and its application.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] In a first aspect, the present invention provides a DNA binding site for the fungal transcription factor AniJ, wherein the sequence of the DNA binding site is RNGCTGAS, wherein R is A or G and S is C or G.

[0007] A second aspect of the invention provides a recombinant expression vector comprising... aniAA gene promoter, wherein at least one DNA binding site as described in this invention is added to the promoter.

[0008] A third aspect of the invention provides a genetically engineered bacterium containing the DNA binding site or recombinant expression vector described in this invention.

[0009] A fourth aspect of the present invention provides a method for constructing genetically engineered bacteria, comprising: introducing at least one DNA binding site as described in the present invention into a promoter of a host cell by mutagenesis or recombination.

[0010] A fifth aspect of the invention provides the use of the DNA binding site, recombinant expression vector, or genetically engineered bacteria described herein in the production of echinocandin B.

[0011] A sixth aspect of the present invention provides a method for producing echinocandin B, comprising:

[0012] (b1) Cultivate the genetically engineered bacteria described in this invention; (b2) Isolate echinococin B from the fermentation product.

[0013] Compared with existing technologies, the above technical solutions have the following advantages:

[0014] (1) This invention identifies the key binding site GAGCTGAC of the transcription factor AniJ, which regulates the biosynthesis of echinocandy, through targeted mutation screening using a dual-base editor, and verifies its specific binding ability using EMSA experiments. Furthermore, a series of enhanced promoters, AnPaniA_e1 (SEQ ID NO: 153), AnPaniA_e2 (SEQ ID NO: 154), and AnPaniA_e3 (SEQ ID NO: 155), were innovatively designed to increase promoter activity by increasing the copy number of the GAGCTGAC motif.

[0015] (2) In the engineered strains aniAe1, aniAe2, and aniAe3 constructed using CRISPR-Cas9 technology in this invention, aniA Gene expression levels were increased by 1.6-3.2 times, and the yield of echinococin B was increased by 1.2-1.6 times compared with the wild type.

[0016] (3) This invention not only provides novel engineered strains and promoter optimization strategies for high production of fungal secondary metabolites, but also provides programmable transcriptional regulatory elements for synthetic biology, which has important application value in the fields of industrial strain modification and synthetic biology. Attached Figure Description

[0017] Figure 1This is a schematic diagram of the method for screening and mining transcription factor AniJ binding sites in an embodiment of the present invention; wherein, A is the echinocandin B biosynthesis gene cluster, and the structural formulas of echinocandin B synthesized by this gene and anidoxane derived from its structure; B is the mutation method using a base editor. aniA A schematic diagram illustrating the process of using the promoter and PyrG-5-FOA screening to discover the binding site of transcription factor AniJ. AniJ is a pathway-specific regulator of the echinocandin B synthesis gene cluster. aniA It is a gene in the gene cluster that encodes nonribosomal peptide synthase; AniJ can activate... aniA In this context, UAS stands for upstream activation sequence.

[0018] Figure 2 As described in the embodiments of the present invention ptH-SpRY-CABE Vector plasmid map;

[0019] Figure 3 The plasmid in the embodiment of the present invention pUC-sgRNA+AnPaniA-Donor Construction and its application in constructing engineered strain JOE-P aniA ::pyrG A schematic diagram;

[0020] Figure 4 Targeted mutation in the embodiments of the present invention aniA sgRNA expression plasmid map of promoter;

[0021] Figure 5 This is a schematic diagram illustrating the distribution of AniJ transcription factor binding sites on the echinocandyne B synthetic gene cluster in an embodiment of the present invention; wherein, A represents the target gene cluster. aniA Following promoter mutation, strains grown on 5-FOA plates were subjected to [further treatment / treatment]. aniA Analysis of promoter region sequencing results; B represents the distribution of the inferred binding sites on the promoters of gene clusters;

[0022] Figure 6 This is a schematic diagram illustrating the in vitro verification of the predicted binding site and the binding of transcription factor AniJ in this embodiment of the invention; wherein, A is a schematic diagram of the purification results of recombinant protein aniJ_DBD-GB1-His and tag control protein GB1-His, and B is a schematic diagram of the DNA binding domain of transcription factor AniJ and... aniA Schematic diagram of in vitro binding verification of the GAGCTGAC motif in the promoter;

[0023] Figure 7 For the JOE strain in the embodiments of the present invention aniA A diagram showing the replacement of the promoter with the AnPaniA_e1 promoter;

[0024] Figure 8Adding to the embodiments of the present invention aniA A schematic diagram illustrating the relationship between the increased number of GAGCTGAC motifs in the promoter and ECB production; where A represents the promoter. AnPaniA_e1 , AnPaniA_e2 , AnPaniA_e3 and AnPaniA_e4 The diagram shows the structure of the promoter. B represents the comparison of transcriptional levels between the engineered promoter and the original promoter. C represents the comparison of echinocandin B production between engineered strains aniAe1, aniAe2, aniAe3, aniAe4 and strain JOE. In the figure, "**" indicates a significant difference compared to strain JOE at p<0.01, "***" indicates a significant difference compared to strain JOE at p<0.01 (p<0.001), and "ns" indicates no significant difference compared to strain JOE. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0027] As mentioned above, in the prior art, aniA The core promoter of the gene and its binding site with the transcription factor AniJ are currently unclear, making it difficult to carry out ECB metabolic pathway modification based on transcriptional regulation optimization. In view of this, this invention provides a DNA binding site for the fungal transcription factor AniJ and its application.

[0028] In one embodiment of the present invention, a DNA binding site for the fungal transcription factor AniJ is provided, characterized in that the sequence of the DNA binding site is RNGCTGAS, wherein R is A or G and S is C or G.

[0029] In some embodiments, the present invention provides a polynucleotide comprising one or more double-stranded DNA binding sites. At least eight bases of the first strand of the site are sequence-identical to 5'-A / GNGCTGAC / G-3'. Since the eight bases in the DNA binding site can be degenerate, only three positions in the sequence may vary.

[0030] In some embodiments, the sequence of the polynucleotide is selected from any of the following:

[0031] (c1) The sequence shown in SEQ ID NO: 153; (c2) The sequence shown in SEQ ID NO: 154; (c3) The sequence shown in SEQ ID NO: 155; (c4) The sequence shown in SEQ ID NO: 156.

[0032] In this invention, the DNA binding site specifically binds to the transcription factor AniJ under binding conditions, and this binding activates the transcription of downstream nucleotide sequences. The DNA binding site can be obtained from eukaryotes (e.g., fungi).

[0033] The DNA binding site of this invention can be obtained from any filamentous fungus. "Filamentous fungus" includes... Eumycota and Oomycota All filamentous forms of filamentous fungi belong to the subphylum. Filamentous fungi are characterized by a hyphal wall composed of chitin, cellulose, dextran, deacetylated chitin, mannan, and other complex polysaccharides. Vegetative growth occurs through hyphal elongation, and carbon metabolism is obligate aerobic. Species of filamentous fungi include, but are not limited to, Acremonium , Aspergillus , Aureobasidium , Cryptococcus , Filibasidium , Fusarium , Humicola , Magnaporthe , Mucor , Myceliophthora , Neocallimastix , Neurospora , Paecilomyces , Penicillium , Piromyces , Schizophyllum , Talaromyces , Thermoascus , Thielavia , Tolypocladium and Trichoderma Those belonging to the genus.

[0034] DNA binding sites are obtained from Aspergillus species, for example A.awamori , A.nidulans , A.niger , A. oryzae , A.sojae or A.pachycristatus Preferably, it comes from... A.nidulans or A.pachycristatus The kind obtained.

[0035] DNA binding sites can be obtained from Aspergillus nidulans ATCC58396 or Aspergillus pachycristatusThe DNA binding site was obtained from ATCC58397. Furthermore, DNA binding sites can be identified and obtained from other sources, including microorganisms isolated from nature (e.g., soil, fertilizer, water, etc.).

[0036] In some embodiments, the DNA binding site is located in the promoter region of the echinocandin B biosynthesis gene cluster, preferably in... aniA The position from -212 bp to -219 bp upstream of the ATG start codon in the gene promoter.

[0037] In some embodiments, the sequence of the DNA binding site is GAGCTGAC.

[0038] In another specific embodiment of the present invention, a method for screening to obtain the DNA binding site of the transcription factor AniJ is provided, comprising: constructing an adenine and cytosine dual-editor expression vector containing the filamentous fungal replication element AMA1 and the hygromycin B resistance gene HygR; constructing aniA Startup driver pyrG Engineered strains of Aspergillus nidus expressing the strain; construction of targeted mutants aniA promoter sgRNA combinatorial library; dual-base editor-based targeted mutation aniA Promoter, explore AniJ binding sites.

[0039] like Figure 1 As shown, this invention identifies the key binding site GAGCTGAC of the transcription factor AniJ, which regulates the biosynthesis of echinocandy, through targeted mutation screening using a dual-base editor, and verifies its specific binding ability using EMSA experiments.

[0040] In another specific embodiment of the present invention, a recombinant expression vector is provided, comprising: aniA A gene promoter in which at least one DNA binding site as described in this invention is inserted, and the binding site regulates the expression of downstream genes.

[0041] In this invention, the regulation is positive regulation.

[0042] In some embodiments of the present invention, the aniA The gene promoter is a modified promoter.

[0043] In some embodiments of the present invention, the insertion location of the DNA binding site can be arbitrary, and those skilled in the art can insert it based on existing technology. aniA Gene promoters increase their copy number to activate the transcription of downstream nucleic acids.

[0044] In some embodiments of the present invention, the number of inserted DNA binding sites can be one or more. When the number of inserted sites is one or more, the DNA binding sites can be connected by a linker sequence to form a multi-copy DNA binding motif based on the linker sequence; or they can be distributed intermittently in the promoter region without being linked by a linker sequence, with the intervening sequence being the original sequence of the promoter region; or the DNA binding sites with modified ends can be inserted into the promoter region with or without a linker sequence, and the modification can be the addition of protective bases or epigenetic modification (such as DNA methylation, phosphorylation, etc.).

[0045] In some embodiments of the present invention, the aniA The gene promoter is AnPaniA_e1, and its nucleotide sequence is shown in SEQ ID NO: 153.

[0046] In some embodiments of the present invention, the aniA The gene promoter is AnPaniA_e2, and its nucleotide sequence is shown in SEQ ID NO: 154.

[0047] In some embodiments of the present invention, the aniA The gene promoter is AnPaniA_e3, and its nucleotide sequence is shown in SEQ ID NO: 155.

[0048] In some embodiments of the present invention, the aniA gene promoter is AnPaniA_e4, and its nucleotide sequence is shown in SEQ ID NO: 156.

[0049] In some embodiments of the present invention, the recombinant expression vector further includes a target natural vector. aniA The sgRNA expression cassette of the gene promoter has the nucleotide sequence shown in SEQ ID NO: 157.

[0050] In some implementations, the recombinant expression vector can be any suitable vector.

[0051] In some implementations, the recombinant expression vector includes, but is not limited to, recombinant cloning vectors, recombinant eukaryotic expression plasmids, or recombinant viral vectors.

[0052] In some implementations, the recombinant eukaryotic expression plasmid includes pCMV , pUC18 , pUC19 , pUC57 , pBAD , pET , pENTR , pGenlenti or pAAV .

[0053] In some implementations, the recombinant expression vector includes pUC19。

[0054] In another specific embodiment of the present invention, a genetically engineered bacterium is provided, which contains the DNA binding site or recombinant expression vector described in the present invention.

[0055] In another specific embodiment of the present invention, a method for constructing the genetically engineered bacteria of the present invention is provided, comprising: introducing at least one DNA binding site of the present invention into the promoter of a host cell through mutagenesis or recombination.

[0056] In some embodiments, the method for constructing the genetically engineered bacteria includes:

[0057] (a1) Design targeting natural aniA (a2) Construct a homologous recombination donor vector containing the modified promoter using the promoter's sgRNA; (a3) ​​Transform the donor vector and the Cas9 expression vector into host cells; (a4) Screen for positive transformants of the modified promoter to obtain the modified promoter; the modified promoter contains at least one DNA binding site as described in this invention, and its nucleotide sequence is shown in any one of SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, and SEQ ID NO: 156.

[0058] In some embodiments, the host cell is Aspergillus nidulans (… Aspergillus nidulans JOE.

[0059] In some embodiments, the strain JOE- P aniA ::pyrG It is by... Aspergillus nidulans JOE strain pyrG gene promoters AnP pyrG Replace with the echinocandin B biosynthesis gene aniA promoter AnP aniA get 。

[0060] In this invention, the construction process of the Aspergillus nidulans JOE strain (also known as strain JOE) has been disclosed in a patent entitled "Genetically Engineered Bacterium with High Echinocandin B Production and Its Application," patent number ZL 202410066032.5. In this strain, aniJ Genes are generated by strong promoters Ptef1 The driver implements overexpression.

[0061] In another specific embodiment of the present invention, the application of the DNA binding site, recombinant expression vector, or genetically engineered bacteria described in the present invention in the production of echinocandin B is provided.

[0062] In some implementations, the application specifically involves increasing the yield of echinocandin B.

[0063] In another specific embodiment of the present invention, a method for producing echinocandin B is provided, comprising:

[0064] (b1) Cultivate the genetically engineered bacteria described in this invention; (b2) Isolate echinococin B from the fermentation product.

[0065] In some embodiments, culturing the genetically engineered bacteria of the present invention specifically includes:

[0066] The spores of the genetically engineered bacteria were inoculated into PDB liquid medium and cultured for 2 days at 24-27℃ and 210-230 rpm to obtain seed culture. The seed culture was then inoculated into fermentation medium and cultured at 24-27℃ and 210-230 rpm to isolate echinocandin B from the fermentation product.

[0067] In some implementations, the seed culture is inoculated into the fermentation medium at a rate of 6% (v / v) for fermentation culture.

[0068] In some implementations, the fermentation culture time is 12 days.

[0069] In some implementations, methyl oleate is added as a supplement on the 5th, 7th and 9th days of fermentation, with the amount added being 2wt% to 4wt%.

[0070] In some implementations, methyl oleate is added as a supplement on the 5th, 7th and 9th days of fermentation, with an addition amount of 3 wt%.

[0071] In some embodiments, the fermentation medium consists of: methyl oleate 100 g / L, glycerol 10 g / L, peptone 40 g / L, K2HPO4·3H2O 8 g / L, MgSO4·7H2O 0.5 g / L, MnSO4·H2O 0.2 g / L, FeSO4·7H2O 0.05 g / L, CaCl2 0.5 g / L, CuSO4·5H2O 0.6 g / L, and pH 7.0.

[0072] Unless otherwise specified, all materials and reagents used in the examples are commercially available.

[0073] Example 1: Construction of an adenine and cytosine dual-editor expression vector containing the filamentous fungal replication element AMA1 and the hygromycin B resistance gene HygR.

[0074] Construct a 17.77 kb long file. ptH-SpRY-CABE The basic structure of the vector plasmid is as follows: Figure 2 As shown: This vector contains filamentous fungal replication elements ( AMA1 Hygromycin B resistance gene ( HygR ), bacterial replication elements ( Ori ), ampicillin resistance gene ( AmpR );in, AMA1 and Ori This vector plasmid can be autonomously replicated in filamentous fungi and bacteria, respectively. HygR and AmpR It can confer resistance to hygromycin B in filamentous fungi and ampicillin resistance in bacteria, respectively; in addition, the vector also contains a gene from Aspergillus nidulans. tfe1 promoter ( AnP tef1 ) and terminator ( AnT tef1 This invention relates to an adenine / cytosine editor expression cassette (SV40-evoCDA1-Tad8e-SpRY-2UGI). The cassette includes a nuclear localization signal peptide (SV40) that guides the editor protein into the nucleus. The nCas9 variant (SpRY) targets the editing site and forms single-strand breaks in DNA under the guidance of sgRNAs with PAMs such as NAN and NGN. Cytosine deaminase (evoCDA1) and adenine deaminase (Tabe8e) convert cytosine C to thymine T and adenine A to guanine G at bases 3 to 10 of the editing target site, respectively. A dual uracil DNA glycosylation inhibitor (2×UGI) protects the cytosine deamination product uracil from degradation. Furthermore, a fungal intron is inserted into the SPRY gene to ensure stable replication of the plasmid in *E. coli*. The vector construction method is as follows:

[0075] Primer design and gene amplification: The coding sequence of the adenine / cytosine editor (SV40-evoCDA1-Tad8e-SpRY-2UGI) synthesized by Genewiz (containing the coding gene for the SV40 nuclear localization signal peptide, the coding gene for the cytosine deaminase evoCDA1, the coding gene for the XTEN linker peptide, the coding gene for the adenine deaminase Tad8e, and the coding sequence for amino acid residues 2 to 92 of the nCas9 variant SpRY, the nucleotide sequence of which is shown in SEQ ID NO: 1) was used to design primer pairs SV40-Ptef1-F (SEQ ID NO: 2) and SV40-Ttef1-R (SEQ ID NO: 3), and the insert fragment (6.37 kb) with the vector backbone linker was amplified using SEQ ID NO: 1 as a template; primer pairs Ttef1-SV40-F (SEQ ID NO: 4) and Ptef1-SV40-R (SEQ ID NO: 5) were also designed and amplified using SEQ ID NO: 1 as a template. pFC332 Using the (Addgene # 87845) vector as a template, amplification was performed containing... AMA1 , HygR , Ori , AmpR , AnP tef1 , AnT tef1 The vector backbone (11.43 kb) included components and insert fragment adapters. The PCR reaction for the above amplification was performed using 2× Phanta UniFiMaster Mix (Nanjing Novizan, P526-03), and the gel extraction and purification of the amplified products were performed using the FastPure GelDNA Extraction Mini Kit (Nanjing Novizan, DC301-01). The PCR reaction and gel extraction / purification conditions were as per the manufacturer's instructions.

[0076] Vector assembly and E. coli transformation: Homologous recombination (i.e., vector assembly) was performed on the gel-purified insert fragment and vector backbone using the ClonExpress Ultra One StepCloning Kit (Nanjing Novizan, C117-02). The assembled product was then transformed into E. coli using Trelief 5α competent cells (Beijing Qingke, TSC-C01). After transformation, the E. coli suspension was plated on LB agar plates containing carbenicillin sodium (100 mg / L) and incubated upside down at 37°C for 16 h. Homologous recombination and E. coli transformation conditions were as per the manufacturer's instructions.

[0077] Identification of E. coli and plasmids in the vector: Single-clonal transformants were picked and resuspended in 10... μIn sterile water, after colony PCR and sequencing verification, a full-length sample of 17.77 kb was finally obtained. ptH-SpRY-ACBE Vector plasmid.

[0078] Example 2: Construction aniA Startup driver pyrG Engineered strains of Aspergillus nidus expressed

[0079] Starting strain Aspergillus nidulans ( Aspergillus nidulans JOE is derived from a patent-pending strain: Genetically engineered bacteria producing high levels of echinocandin B and its applications, patent number ZL 202410066032.5. In this engineered strain, aniJ Genes are generated by strong promoters Ptef1 The driver implements overexpression.

[0080] like Figure 3 As shown, based on the CRISPR / Cas9 system and homology-directed repair strategy, Aspergillus nidulans JOE strain... pyrG Gene promoters ( AnP pyrG Replace with the echinocandin B biosynthesis gene. aniA promoter ( AnP aniA ), to obtain engineered strain JOE- P aniA ::pyrG Because AniJ is the driver aniA Gene expression-specific transcription factors, therefore engineered strain JOE- P aniA ::pyrG In pyrG Genes can aniA Overexpression driven by the promoter and transcription factor AniJ. The specific construction method is as follows:

[0081] Step 1: Construct a dual-function vector for sgRNA expression and recombinant donor.

[0082] Construct a 4.77 kb long file. pUC-sgRNA+AnP aniA -Donor The vector plasmid contains bacterial replication elements ( Ori ), ampicillin resistance gene ( AmpR ), targeted AnP pyrG sgRNA expression cassette at the site, and a segment used to express sgRNA at the site. AnP pyrG Replace with An PaniA The donor DNA fragment; wherein, the sgRNA expression cassette is expressed by a tRNA promoter and contains AnPpyrG The gRNA targeting sequence at the site (located at positions 291 to 310 of the expression cassette sequence); while the donor fragment is... AnP pyrG Upstream / downstream homologous arms ( AnP pyrG _Up / AnP pyrG _Dn) and the promoter sandwiched between them AnP aniA Composition, can be AnP pyrG After the site is cut by Cas9, it is repaired in a targeted manner to ultimately achieve... AnP aniA right AnP pyrG Precise replacement.

[0083] The construction method is as follows: the target was synthesized by Genewiz. AnP pyrG The sgRNA expression cassette sequence of the site (SEQ ID NO: 6) was used to design primer pairs Target_F1-Ori-F (SEQ ID NO: 7) and Target_F2-AmpR-R (SEQ ID NO: 8), and using SEQ ID NO: 6 as a template, an assembly fragment (0.51 kb) with assembly adapters was amplified. Primer pairs AnPpyrG_Up-pUC_Ori-F (SEQ ID NO: 9) and AnPpyrG_Up-AnPaniA-R (SEQ ID NO: 10), AnPpyrG_Dn-AnPaniA-F (SEQ ID NO: 11) and AnPpyrG_Dn-pUC_AmpR-R (SEQ ID NO: 12), and AnPaniA-AnPpyrG_Up-F (SEQ ID NO: 13) and AnPaniA-AnPpyrG_Dn-R (SEQ ID NO: 8) were also designed. NO: 14), and amplified the assembly adapters from the gDNA of Aspergillus nidulans strain JOE. AnP pyrG _Up (1.03 kb) AnP pyrG _Dn (1.03 kb) and AnP aniA_WT(0.54 kb) assembled fragment; primer pairs pUC_AmpR-F (SEQ ID NO: 15) and pUC_AmpR-Target_F2-R (SEQ ID NO: 16) and primer pairs pUC_Ori-Target_F1-F (SEQ ID NO: 17) and pUC_Ori-R (SEQ ID NO: 18) were designed, and from pUC19 The vector was used to amplify cells with assembly connectors. AmpR (1.63kb) and Ori (0.76 kb) element fragment; after recovering the above fragment, it was recombined into 6 fragments using the ClonExpress UltraOne Step Cloning Kit, and the recombinant product was transformed into E. coli and the transformants were identified, finally obtaining pUC-sgRNA+An PaniA -Donor Vector plasmid.

[0084] Step 2: JOE transformation of Aspergillus nidulans and identification of transformants.

[0085] (1) Protoplast transformation of Aspergillus nidulans JOE.

[0086] Transformation was performed on Aspergillus nidulans JOE strain. Frozen Aspergillus nidulans glycerol strain was plated on GMM agar plates and cultured for 5 days. Spores were scraped from the plates and transferred to 50 mL of GMM liquid medium. The culture was carried out at 28°C and 200 rpm for 12 h. After centrifugation (10,000 g, 10 min, room temperature), all supernatant was discarded. The mycelium was placed in 30 mL of enzymatic digestion buffer (containing 1.2 g VinoTaste Pro and 0.09 g Yatalase) and digested at 30°C and 90 rpm for 6 h. 10 mL of the digestion product was transferred to a 15 mL centrifuge tube, and 4 mL of pre-chilled Trapping Buffer (containing 0.6 M Sorbitol, 0.1 M Tris-HCl, pH 7.0) was slowly added. The tube was then centrifuged (2,430 g, 2 min, 4°C). The protoplasts in the intercalation layer were transferred to a 50 mL centrifuge tube using a 1 mL pipette tip, and 3 times the volume of pre-chilled STC Buffer (containing 1.2 g Sorbitol, 0.1 M Tris-HCl, pH 7.0) was added. The protoplasts were prepared with 0.01 M Sorbitol, 0.01 M CaCl2, and 0.01 M Tris-HCl (pH 7.5), followed by centrifugation (2,430 g, 2 min, 4 °C) and discarding the supernatant. The protoplasts were resuspended in 0.1–0.5 mL of pre-chilled STC Buffer and diluted to 10⁻⁶. 7 CFU / mL; Add 5 μg of protoplast suspension to 0.05 mL of the protoplast suspension. pFC332Vector plasmid (Addgene # 87845) and 5 μg pUC-AnP pyrG -sgRNA+AnP aniA -Donor The vector plasmid was mixed with DNA and slowly stirred with a 0.2 mL pipette tip, then placed on ice for 3 h. 0.6 mL of PEG solution (containing 60% PEG4000, 0.05 M CaCl2, 0.05 M Tris-HCl, pH=7.5) was added and gently pipetted to mix, then incubated at room temperature for 20 min. The transformation product was plated on GMM (containing 1.2 M mannitol and 100 mg / L hygromycin B, 100 mg / L uridine and 100 mg / L uracil) selection plates and incubated at 30 °C for 4 days until colonies grew.

[0087] The GMM basal medium formulation is as follows: 10 g / L glucose, 20 mM ammonium nitrate, 0.52 g / L KCl, 0.52 g / L MgSO4•7H2O, 1.52 g / L KH2PO4, 1 mL trace elements, pH=6.5. The trace element composition (per 100 mL) is as follows: 2.20 g ZnSO4•7H2O, 1.10 g H3BO3, 0.50 g MnCl2•4H2O, 0.16 g FeSO4•7H2O, 0.16 g CoCl2•5H2O, 0.16 g CuSO4•5H2O, 0.11 g (NH4)6Mo7O 24 • 4H2O, 5.00 g Na4EDTA.

[0088] (2) Identification of Aspergillus nidulans transformants.

[0089] The transformants were identified. Single-clonal transformants were picked and inoculated into fresh PDA liquid medium (containing 100 mg / L uridine and 100 mg / L uracil) and cultured at 28°C and 200 rpm for 2 days. Mycelia were collected by centrifugation (10,000 g, 10 min, room temperature), and total DNA was extracted using a fungal genomic DNA rapid extraction kit (Shanghai Sangon Biotech, B518229). Primer pairs AnPpyrG_Up-Ce-F (SEQ ID NO: 19) and AnPpyrG_Dn-pUC_AmpR-R (SEQ ID NO: 12) were used to extract DNA. P aniA :: pyrGThe site was identified by PCR. If only a 1585 bp band was amplified, it was a homozygous positive clone. If only a 1319 bp band was amplified, it was a homozygous wild type. The PCR products of the homozygous positive clones were sequenced to further confirm whether their sequences were consistent with the donor sequences of the corresponding vectors.

[0090] Plasmid removal was performed on positive transformants. The positive transformant strains were subjected to at least three cycles of PDA (100 mg / L uridine and 100 mg / L uracil) liquid culture for transformation, and the Cas9 expression plasmid was then transferred. pFC332 Remove. At this point, preserve the strain with glycerol for later use.

[0091] Example 3: Constructing Targeted Mutants aniA promoter sgRNA combinatorial library

[0092] Build targeted aniA promoter (i.e.) AnP aniA A library of sgRNA expression vector plasmids containing the complete sequence of sgRNA, which is designed to target specific RNAs. AnP aniA All adenine (A) and cytosine (C) bases in the positive chain pUC-AnP aniA -Target_F01 to pUC-AnP aniA -Target_F63 And all adenine (A) and cytosine (C) bases in the antisense strand pUC-AnP aniA - Target_R01 to pUC-AnP aniA -Target_R60 The vector consists of 123 plasmids. pUC-AnP aniA -Target The basic structure of the carrier (2.29 kb in total length) is as follows: Figure 4 As shown: This vector contains bacterial replication elements ( Ori ), ampicillin resistance gene ( AmpR ), and targeted AnP aniA sgRNA expression cassettes at different sites. pUC-AnP aniA -Target The method for constructing the carrier is as follows:

[0093] Step 1: Primer design and gene amplification.

[0094] based on AnP aniAThe distribution of A and C bases in the sequence and the characteristics of the adenine / cytosine editor's editing window (3-9 bp) are important factors for understanding the relationship between these factors and the overall structure of the sequence. AnP aniA Design sgRNA sequences with positive / antisense strands to target An PaniA The sgRNA sequences of the A and C bases in the sense strand are shown in SEQ ID NO: 20 to SEQ ID NO: 82, targeting... An PaniA The sgRNA sequences of bases A and C in the antisense strand are shown in SEQ ID NO: 83 to SEQ ID NO: 142. Referring to the sgRNA expression cassette sequence in Example 2, the sgRNA sequence in SEQ ID NO: 6 (located at positions 291 to 310 of SEQ ID NO: 6) is replaced with a single target. AnP aniA The sgRNA sequences of the positive and antisense strands (as shown in SEQ ID NO: 20 to SEQ ID NO: 142, respectively) were synthesized by Genewiz, and the corresponding sgRNA expression cassette sequences were synthesized (the vector corresponding to the positive strand sgRNA expression cassette was...). pUC-AnP aniA -Target_F01 to pUC-AnP aniA -Target_ F63 The vector corresponding to the antisense strand sgRNA expression cassette is pUC-AnP aniA -Target_R01 to pUC-AnP aniA -Target_R60 Primer pairs Target_F1-AmpR-F (SEQ ID NO: 143) and Target_F2-Ori-R (SEQ ID NO: 144) were designed, and assembly fragments (0.54 kb) with vector adapters were amplified from SEQ ID NO: 20 to SEQ ID NO: 142, respectively; primer pairs pUC_AmpR-F (SEQ ID NO: 145) and pUC_Ori-R (SEQ ID NO: 146) were used, and from... pUC19 A vector backbone containing AmpR and Ori elements (1.79 kb) was amplified on the vector.

[0095] Step 2: Vector assembly and E. coli transformation.

[0096] After recovering the above fragments, the vector backbone was recombined with each assembled fragment using the ClonExpress Ultra One Step Cloning Kit. The recombinant products were then transformed into *E. coli* and the transformants were identified. Finally, the following results were obtained: pUC-AnP aniA -Target_F01 to pUC-AnP aniA -Target_F63 as well as pUC- AnP aniA -Target_R01 to pUC-AnP aniA -Target_R60 Vector plasmids (a total of 123).

[0097] Step 3: Preparation of sgRNA combinatorial library.

[0098] The plasmid concentration of each sgRNA expression vector was adjusted to 100 ng / mL using sterile water. μ L, then take 2 μ The various plasmid solutions of L were mixed to obtain the final product for targeting. AnP aniA A library of sgRNA expression vector plasmids containing all A and C bases in the sense and antisense strands.

[0099] Example 4: Targeted Mutation Based on a Two-Base Editor aniA Promoter, exploring AniJ binding sites

[0100] The construction of Example 1 ptH-SpRY-CABE Transformed the engineered strain JOE- obtained in Example 2 with the vector and the sgNRA combinatorial library constructed in Example 3. P aniA :: pyrG to it aniA The promoter undergoes full-coverage targeted mutation. If aniA A mutation at the AniJ binding site of the promoter will prevent AniJ from recognizing the promoter, thus hindering downstream... pyrG Gene expression silencing. Due to pyrG The encoded nucleoside-5'-phosphate decarboxylase can convert non-toxic 5-FOA into toxic 5-fluorouracil, thus allowing for reverse screening using 5-FOA medium: strains that can grow on 5-FOA-containing plates indicate that their AniJ binding sites may be inactivated. Potential AniJ binding sites can be identified by screening positive clones and performing sequencing analysis.

[0101] The specific operating steps are as follows:

[0102] Step 1:pyrG Construction of expression silence clones.

[0103] With JOE- P aniA ::pyrG For the starting strain, protoplasts were prepared using the method described in Example 1; 0.05 mL of protoplast suspension was added to... ptH-SpRY-CABE Mixed DNA of vector plasmid and sgRNA combined library plasmid (5 each) μ g), and then the DNA was transformed into protoplasts using PEG transformation; the transformation product was plated on GMM (containing 100 mg / L uridine and uracil, 1.2 M sorbitol and 100 mg / L hygromycin B) selection plates and incubated at 30°C for 5 days.

[0104] Step 2: Sequencing and Identification aniA The core region of the promoter and the AniJ binding site.

[0105] The transformed strains were subjected to genomic extraction and PCR identification. Total DNA was extracted using a fungal genomic DNA rapid extraction kit (Shanghai Sangon Biotech, B518229); primer pairs AnPpyrG_Up-Ce-F (SEQ ID NO: 19) and AnPpyrG_Dn-pUC_AmpR-R (SEQ ID NO: 12) were used to identify each transformant. aniA PCR amplification was performed using the promoter; the amplified products identified by PCR were identified using AnPpyrG_Up-Ce-F primers (SEQ ID NO: 19), and the obtained sequences were then analyzed. aniA Differences in promoter sequences (i.e., mutation sites); sequencing results as follows Figure 5 As shown in A, the mutation frequency is highest at the position -212 bp to -219 bp upstream of ATG (GAGCTGAC), meaning this position is... aniA The core region of the promoter and potential AniJ binding sites were identified. Subsequently, we analyzed the distribution of the predicted AniJ binding sites in the echinocandin B gene cluster using bioinformatics methods. aniA / B / C / D / E / F / F2 / G / H / I / K The promoter sequences were submitted to the MEME 5.5.8 online software (https: / / meme-suite.org / meme) to predict the gene regulatory elements (motifs) within them. The motif analysis mode, distribution mode, length, and number were selected as "Classic mode," "any number of repetitions," "4-10 bp," and "20," respectively. Analysis results showed that the RNGCGGAS motif was present in all of the above promoters, consistent with the sequencing mutation sequence. Figure 5 As shown in B, this motif is inaniA / B / C / D / E / F / F2 / G / H / I / K The promoters all have different numbers of these components.

[0106] Example 5: In vitro validation of predicted binding sites to transcription factor AniJ

[0107] Example 4 shows that, aniA The GAGCGTGAC motif in the promoter is a potential binding site for AniJ. This embodiment uses electrophoretic mobility shift assay (EMSA) to verify the binding of this motif to the transcription factor AniJ. A recombinant protein (His-MBP-aniJ_DBD) with a His-MBP tag containing the DNA-binding domain of the transcription factor AniJ was compared with a truncated protein containing GAGCGTGAC and labeled with 5'-IR700 fluorescence. aniA The promoter probe (200 bp to -229 bp upstream of ATG) was co-incubated in vitro, and the incubation products were separated by electrophoresis using a non-denaturing TBE-PAGE gel. The DNA-protein complex was then detected by IR700 fluorescence signal to verify the presence of Aspergillus nidulans. aniA The binding of this motif to the transcription factor AniJ in the promoter is as follows: The steps are as follows:

[0108] Step 1: Preparation of recombinant protein and DNA probe.

[0109] Preparation of recombinant proteins: Recombinant proteins (aniJ_DBD-GB1-His, amino acid sequence shown in SEQ ID NO: 147) containing the His and GB1 tags and the aniJ DNA-binding domain were expressed and purified by GenScript. A control protein without the aniJ_DBD tag (GB1-His, amino acid sequence shown in SEQ ID NO: 148) was also produced. The protein purification results are as follows: Figure 6 As shown in A in the diagram.

[0110] Preparation of DNA probes: Truncated DNA probes containing GAGCTGAC and modified with 5'-IR700 fluorescence were synthesized by Genewiz. aniAThe positive and negative oligonucleotides (SEQ ID NO: 149 and SEQ ID NO: 150) of the promoter (upstream of ATG -200 bp to -229 bp) were synthesized. Simultaneously, oligonucleotides without fluorescent modification, such as those shown in SEQ ID NO: 149 and SEQ ID NO: 150, as well as the motif mutant positive oligonucleotides (SEQ ID NO: 151 and SEQ ID NO: 152) of the truncated promoter were synthesized. The fluorescently modified SEQ ID NO: 149 and SEQ ID NO: 150, the unfluorescently modified SEQ ID NO: 149 and SEQ ID NO: 150, and the SEQ ID NO: 151 and SEQ ID NO: 152 were mixed in a 1:1 ratio and incubated in a PCR instrument at 95°C for 10 min, then cooled to room temperature for later use. The three sets of probes prepared were hot probes, cold probes, and mutant probes, respectively.

[0111] Step 2: Preparation of DNA-protein complex.

[0112] After thawing the corresponding protein, probe, and EMSA binding buffer (containing 5 mM dithiothreitol, 2.5 mM EDTA, 0.25% Triton X-100, 25% glycerol, 0.1 M Tris-HCl, pH=8.0) on ice, prepare the DNA-protein binding system on ice according to the following parameters.

[0113] Table 1. Preparation of DNA-protein binding reaction system

[0114]

[0115] After incubating the prepared binding system at 30°C for 20 min, add EMSA loading buffer (Shanghai Beyotime, GS007) for loading.

[0116] Step 3: Electrophoretic detection of DNA-protein complexes.

[0117] Pre-electrophoresis: Load the TBE-PAGE electrophoresis gel (Shanghai Beyotime, D0171S) into a vertical electrophoresis tank (Beijing Junyi, JY-SCZ2+); dilute 5×TBE buffer (Shanghai Beyotime, ST719) to 0.5×TBE electrophoresis solution and inject it into the electrophoresis tank. Perform pre-electrophoresis at 100 V for 60 min to observe whether the voltage is normal; after the pre-electrophoresis is completed, turn off the power and rinse the sample wells with electrophoresis solution.

[0118] Sample loading and electrophoresis: Load all samples containing EMSA loading buffer into the corresponding wells, and fill each well with an equal volume of 1×EMSA loading buffer; perform electrophoresis at 100 V and stop electrophoresis when the bromophenol blue dye migrates to 2 / 3 of the gel.

[0119] Imaging and Analysis: The gel was removed from the electrophoresis tank and placed in a near-infrared laser imaging system (LI-CORBiosciences, Odyssey DLx, USA) and imaged using IR700 mode; the results are as follows. Figure 6 As shown in B, interaction group 1 contains a DNA-protein complex band, indicating that the hot probe (i.e., the IR700-labeled truncated probe) is present. aniA The promoter fragment can bind to the aniJ_DBD-GB1-His protein to form a DNA-protein complex; no DNA-protein complex band was observed in controls 1 and 2, indicating that the binding of the hot probe to the aniJ_DBD-GB1-His protein is a specific binding mediated by the transcription factor AniJ DNA-binding domain (aniJ_DBD); due to the competitive effect of the cold probe, the DNA-protein complex band signals in groups 1 and 2 were weakened or even disappeared, further proving that aniJ_DBD is associated with truncated DNA-protein complexes. aniA The specificity of promoter fragment binding; because the GAGCGTGAC motif in the mutant probe has been mutated, the mutant probe cannot compete with the hot probe in the DNA-protein complex, resulting in no significant attenuation of the DNA-protein complex band signal in the three competing groups, indicating that truncation... aniA The GAGCTGAC in the promoter can bind to aniJ_DBD in vitro.

[0120] Example 6: Add aniA The number of GAGCTGAC motifs in the promoter increases transcriptional levels and ECB production.

[0121] This embodiment increases the *Aspergillus nidus* JOE strain. aniA The number of GAGCTGAC motifs in the promoter, and subsequently the construction of a system driven by promoters with different modifications. aniA Engineered strains of Aspergillus nidulans expressing genes; subsequently, the performance of each engineered strain was evaluated. aniA Gene expression levels and the yield of the product echinocandin B. The specific operational steps are as follows:

[0122] Step 1: Build the project starter driver with the added GAGCTGAC sequence aniA Engineered strains of Aspergillus nidus expressed

[0123] Using CRISPR / Cas9-mediated homologous recombination, the homologous recombination of strain JOE was... AnPaniA Replace with AnPaniA_e1 to AnPaniA_e4 (in AnPaniA Replace with AnPaniA_e1 The schematic diagram is as follows Figure 7 As shown), to obtain the corresponding starter driver. aniA The engineered strains aniAe1, aniAe2, aniAe3, and aniAe4 were expressed, among which the promoter... AnPaniA_e1 , AnPaniA_e2 , AnPaniA_e3 and ​ Structural diagram as follows ​ As shown in A in the diagram.

[0124] The above ​ The promoter is in ​ A GAGCTGAC motif is inserted 156 bp upstream of the ATG promoter, and the sequence is shown in SEQ ID NO: 153.

[0125] The above ​ The promoter is in ​ A GAGCTGAC motif is inserted 180 bp upstream of the ATG promoter, and the sequence is shown in SEQ ID NO: 154.

[0126] The above ​ The promoter is in ​ The GAGCGTGAC motif is inserted at -156 bp and -180 bp upstream of the ATG promoter, and the sequence is shown in SEQ ID NO: 155.

[0127] The above ​ The promoter is in ​ The GAGCGTGAC motif is inserted 273 bp upstream of the ATG promoter, and the sequence is shown in SEQ ID NO: 156.

[0128] The specific method is as follows:

[0129] (1) Construct a dual-function vector for sgRNA expression and recombinant donor.

[0130] Construct a 4.64kb file. ​ aniA_e1 ​ The basic structure of the vector plasmid is as follows: ​ As shown: This vector contains bacterial replication elements ( ​ ), ampicillin resistance gene ( ​ ), targeted ​ aniA Edit site 1 sgRNA expression cassette, and a segment for editing ... ​ aniA_WT Replace with ​aniA_e1 The donor DNA; wherein, the sgRNA expression cassette is expressed by a tRNA promoter and contains ​ aniA Editing site 1 gRNA target sequence; while the donor DNA is from ​ aniA Upstream / downstream homologous arms ( ​ aniA ​ / ​ aniA ​ ) and those sandwiched between the two ​ aniA_e1 Composition, can be ​ aniA_WT After the site is cut by Cas9, it is repaired in a targeted manner to ultimately achieve... ​ aniA_e1 right ​ aniA_WT Precise replacement. The construction method is as follows: Design target ​ aniA The sgRNA at editing site 1 (SEQ ID NO: 157) was modified by replacing the sgRNA sequence in SEQ ID NO: 6 (located at positions 291 to 310 of SEQ ID NO: 6) with the sequence shown in SEQ ID NO: 157, according to the sgRNA expression cassette sequence in Example 2. The corresponding sgRNA expression cassette sequence was synthesized by Genewiz. Using primer pairs Target_F1-Ori-F (SEQ ID NO: 7) and Target_F2-AmpR-R (SEQ ID NO: 8), an assembly fragment (0.51 kb) with an assembly adapter was amplified using SEQ ID NO: 157 as a template. Using primer pairs SEQ ID NO: 158 and SEQ ID NO: 159, and primer pairs SEQ ID NO: 160 and SEQ ID NO: 161, and using gDNA from Aspergillus nidulans strain JOE as a template, fragments with assembly adapters were amplified respectively. ​ aniA ​ (0.96 kb) and ​ aniA ​ (0.94 kb) assembled fragment; synthesized by Genewiz with the GAGCGTGAC motif added at a position 156 bp upstream of ATG. ​ aniA_e1The sequence (SEQ ID NO: 153) was amplified using primer pairs SEQ ID NO: 162 and SEQ ID NO: 163 to produce a fragment (0.56 kb) with an assembly adapter; primer pairs pUC_AmpR-F (SEQ ID NO: 15) and pUC_AmpR-Target_F2-R (SEQ ID NO: 16) and pUC_Ori-Target_F1-F (SEQ ID NO: 17) and pUC_Ori-R (SEQ ID NO: 18) were used, and... ​ Using the vector as a template, amplification was performed using vectors with assembly adapters. ​ (1.63 kb) and ​ (0.76 kb) element fragments; after recovering the above fragments, the above 6 fragments (sgRNA expression cassette, ...) were processed using the ClonExpress Ultra One Step Cloning Kit homologous recombination kit. ​ aniA ​ aniA_e1 ​ aniA ​ and ​ The recombinant product was recombined and assembled, and then transformed into E. coli and the transformants were identified to obtain the final product. ​ aniA_e1 ​ Vector plasmid.

[0131] Taking this as an example, construct ​ aniA ​ Plasmid. Using primer pair SEQ ID NO: 162 and SEQ ID NO: 163, to ​ aniA_e2 Using the sequence (SEQ ID NO: 154) as a template, the GAGCGTGAC motif was added 180 bp upstream of ATG. ​ aniA_e2 Sequences. The above 6 fragments (sgRNA expression cassette, ...) will be processed using the ClonExpress Ultra One Step Cloning Kit homologous recombination kit. ​ aniA ​ aniA_e2 ​ aniA ​ and ​ The recombinant product was recombined and assembled, and then transformed into E. coli and the transformants were identified to obtain the final product. ​ aniA_e2 -​ Vector plasmid.

[0132] Taking this as an example, construct ​ aniA ​ Plasmid. Using primer pair SEQ ID NO: 162 and SEQ ID NO: 163, to ​ aniA_e3 Using the sequence (SEQ ID NO: 155) as a template, the GAGCGTGAC motif was added at -156 bp and -180 bp upstream of ATG. ​ aniA_e3 Sequences. The above 6 fragments (sgRNA expression cassette, ...) will be processed using the ClonExpress Ultra OneStep Cloning Kit homologous recombination kit. ​ aniA ​ aniA_e3 ​ aniA ​ and ​ The recombinant product was recombined and assembled, and then transformed into E. coli and the transformants were identified to obtain the final product. ​ ​ aniA_e3 ​ Vector plasmid.

[0133] Taking this as an example, construct ​ aniA ​ Plasmid. Using primer pair SEQ ID NO: 162 and SEQ ID NO: 163, to AnP aniA_e4 Using the sequence (SEQ ID NO: 156) as a template, the GAGCGTGAC motif was added at a position 273 bp upstream of ATG. AnP aniA_e4 Sequences. The above 6 fragments (sgRNA expression cassette, ...) will be processed using the ClonExpress Ultra One Step Cloning Kit homologous recombination kit. AnP aniA _Up、AnP aniA_e4 、AnP aniA _Dn、AmpR and Ori The recombinant product was recombined and assembled, and then transformed into E. coli and the transformants were identified to obtain the final product. pUC-SgRNA+AnP aniA_e4 - Donor Vector plasmid.

[0134] (2) Transformation of Aspergillus nidulans and identification of transformants.

[0135] Following the protoplast preparation method in Example 1, 0.05 mL of Aspergillus nidulans JOE strain protoplast suspension was added to mixed plasmid DNA (5 μg each), and then the DNA was transformed into protoplasts using the PEG transformation method. The transformation product was plated on GMM (1.2 M sorbitol and 100 mg / L hygromycin B) selection plates and cultured at 30°C for 4 days.

[0136] The transformations were divided into four groups. The first group consisted of mixed plasmids including the Cas9 expression plasmid pFC332 (Addgene # 87845) and... pUC-sgRNA+AnP aniA_e1 -Donor , used to AnP aniA_e1 Replace the original AnP aniA The obtained strain was named aniAe1.

[0137] The second group of mixed plasmids includes the Cas9 expression plasmid. pFC332 (Addgene # 87845) and pUC sgRNA+ AnP aniA_e2 -Donor , used to AnP aniA_e2 Replace the original AnP aniA The obtained strain was named aniAe2.

[0138] The third group of mixed plasmids includes Cas9 expression plasmids. pFC332 (Addgene # 87845) and pUC-sgRNA+ AnP aniA_e3 -Donor , used to AnP aniA_e3 Replace the original AnP aniA The obtained strain was named aniAe3.

[0139] The fourth group of mixed plasmids includes the Cas9 expression plasmid. pFC332 (Addgene # 87845) and pUC-sgRNA+ AnP aniA_e4 -Donor , used to AnP aniA_e4 Replace the original AnP aniA The obtained strain was named aniAe4.

[0140] The transformants were subjected to DNA extraction and PCR identification, and the PCR products of positive clones were sequenced for identification.

[0141] Step 2: Quantitative verification of the addition of GAGCTGAC motif pairs aniA The impact of expression levels.

[0142] The three strains (aniAe1, aniAe2, aniAe3, and aniAe4) and the JOE strain from step one were inoculated into GMM liquid medium and cultured at 25°C and 200 rpm for 2 days. Mycelia were collected by centrifugation (10,000 g, 10 min, room temperature). Total RNA was then extracted and reverse transcribed from the mycelia of each strain using a column-based fungal total RNA extraction and purification kit (Shanghai Sangon Biotech, B518659) and a reverse transcription kit (HiScript III RT SuperMix for qPCR (+gDNA wiper)) (Nanjing Novizan, R323-01). *Aspergillus nidulans* was used as the starting material for the reverse transcription. Actin The gene was used as an internal reference gene (primers were SEQ ID NO: 164 and SEQ ID NO: 165). The SupRealQ Ultra Hunter SYBR qPCR Master Mix (U+) qPCR kit (Nanjing Novizan, Q713-03) was used to detect the genes in each group of strains. aniA Quantitative detection of gene transcription levels was performed (primers: SEQ ID NO: 166 and SEQ ID NO: 167); subsequently, using strain JOE as a control, 2 -ΔΔCt The algorithm for each experimental group of strains aniA Gene transcription and expression levels are calculated.

[0143] like Figure 8 As shown in B, strains aniAe1, aniAe2, aniAe3, and aniAe4... aniA The transcriptional expression levels were 1.8-fold, 1.6-fold, 3.2-fold, and 0.98-fold higher than those of the control group, indicating an increase in expression. aniA The copy number of GAGCTGAC in the promoter can be increased. aniA Gene expression, and aniA Gene expression levels were correlated with the copy number of GAGCGTGAC. However, expression levels were not significantly increased in the aniAe4 strain, indicating that the specific insertion location of this element may affect its regulatory effect, and optimization is needed to fully realize its synergistic effect.

[0144] (2) Verification PaniA The effect of promoter optimization on the yield of echinocandin B.

[0145] The four strains (aniAe1, aniAe2, aniAe3, and aniAe4) from step one, as well as strain JOE, were subjected to fed-batch fermentation in shake flasks. Spores from these strains were inoculated into 30 mL of PDB liquid medium (Solepro, P9240) and cultured at 25°C and 220 rpm for 2 days. Then, 3 mL of the seed culture was inoculated into 50 mL of fermentation medium and cultured for another 12 days at 25°C and 220 rpm. The fermentation medium consisted of: methyl oleate 100 g / L, glycerol 10 g / L, peptone 40 g / L, K₂HPO₄·3H₂O 8 g / L, MgSO₄·7H₂O 0.5 g / L, MnSO₄·H₂O 0.2 g / L, FeSO₄·7H₂O 0.05 g / L, CaCl₂ 0.5 g / L, CuSO₄·5H₂O 0.6 g / L, and pH 7.0. On days 5, 7, and 9 of fermentation, 1.5 g of methyl oleate was added to the feed. Fermentation was completed after 12 days.

[0146] Echinocandin B was detected and quantified using ultraviolet-high performance liquid chromatography (UV-HPLC), with an injection volume of 10. μ L. The sample was eluted using a binary gradient elution system consisting of (A) methanol and (B) water (containing 0.1% formic acid, v / v). The elution gradient was: 0–5 min, 5% A; 5–20 min, 5%–95% A; 20–25 min, 95% A; 25–30 min, 5% A; the flow rate was 1.0 mL / min. Metabolites were separated using a Shim-pack GIST C18 column (Shimadzu, Japan, 227-30017-07) at 30 °C, and the chromatograms were monitored by UV absorbance at 210 nm. Echinocandin B standard (Santa Cruz Biotechnology) was used as a standard reference, and quantification of echinocandin B was based on the peak area obtained from HPLC analysis.

[0147] like Figure 8 As shown in Figure C, the echinocandin B production of strains aniAe1, aniAe2, and aniAe3 was 1.2 times, 1.2 times, and 1.6 times that of the control group, respectively, indicating that increased echinocandin production... aniA The copy number of the GAGCTGAC motif in the promoter can increase the yield of echinocandin B. However, the yield of echinocandin B in the aniAe4 strain did not show a significant increase, which is consistent with the qPCR results.

[0148] It should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A modified promoter of the fungal transcription factor AniJ, characterized in that, The nucleotide sequence of the modified promoter is shown in any one of SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO:

155.

2. A recombinant expression vector, characterized in that, It comprises at least one modified promoter as claimed in claim 1.

3. The recombinant expression vector of claim 2, wherein, The recombinant expression vector comprises pUC19.

4. Genetically engineered bacteria, characterized in that, It comprises the modified promoter as claimed in claim 1 or the recombinant expression vector as claimed in any one of claims 2-3.

5. The method for constructing the genetically engineered bacteria according to claim 4, characterized in that, Comprising: Introducing the modified promoter as claimed in claim 1 into a host cell by genetic recombination.

6. The construction method of claim 5, wherein, Comprising: (a1) designing sgRNAs targeting the native aniA promoter, constructing homologous recombination donor vectors comprising the engineered promoters; (a2) transforming the donor vector and the Cas9 expression vector into a host cell; (a3) screening positive transformants to obtain a modified promoter, i.e. The nucleotide sequence of the modified promoter is shown in any one of SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO:

155.

7. The construction method of any one of claims 5-6, wherein, The host cell is Aspergillus nidulans Aspergillus nidulans ) JOE 。 8. The use of the modified promoter of claim 1 or the recombinant expression vector of any one of claims 2-3 or the genetically engineered bacteria of claim 4 in the production of echinocandins B, characterized in that, The genetically engineered strain is Aspergillus nidulans JOE strain.

9. A process for the production of echinocandins B, characterized in that, Comprising: (b1) culturing the genetically engineered strain obtained by the construction method as claimed in claim 6; the genetically engineered strain is Aspergillus nidulans JOE strain; (b2) isolating echinocandin B from the fermentation product.

10. The method of claim 9, wherein, The method further comprises feeding methyl oleate during the culturing process.

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