Genetically engineered bacteria with high production of echinocandin B and its application

Abstract

The present invention discloses a genetically engineered bacterium with high yield of echinocandin B and its application, wherein the genetically engineered bacterium overexpresses the aniJ gene or overexpresses the ecdJ gene, and the starting bacterium of the genetically engineered bacterium is Aspergillus. The present invention also provides a construction method of the genetically engineered bacterium, using a homologous recombination method, using a glufosinate resistance gene bar as a screening gene, and using a strong promoter of Aspergillus nidulans to overexpress the gene in the echinocandin B biosynthesis gene cluster of the strain to obtain a recombinant bacterium. The echinocandin B yields of the two strains of genetically engineered bacteria obtained by the present invention are 10.2 times and 10.6 times that of wild bacteria, respectively, which greatly improves the yield of the target product.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to genetic engineering bacteria with high production of echinocandin B and application thereof. Background Art

[0002] Anidulafungin is a lipopeptide antifungal drug derived from filamentous fungi. It was launched in Europe and the United States by Pfizer in 2007, with annual sales of US$133 million (2019). This type of echinocandin drug can non-competitively inhibit β-1,3-glucose synthase, hinder the synthesis of pathogenic fungal cell walls, and inhibit fungal growth. In recent years, the number of invasive fungal infections complicated and secondary to the new coronavirus has surged, leading to a rapid growth in the market for antifungal drugs such as anidulafungin, and the global market is in short supply. However, the production process of anidulafungin includes two complex processes: fermentation production of the precursor echinocandin B (ECB) and chemical post-modification, and the price for clinical use remains high. At present, the optimization of production strains is still mainly based on mutagenesis breeding. Due to the unstable fermentation yield of mutagenic strains and the large number of by-products, the production price of raw materials is high, which restricts the large-scale production and clinical application of anidulafungin. Summary of the invention

[0003] In order to solve the above problems in the prior art, the present invention provides a genetically engineered bacterium with high production of echinocandin B and its application.

[0004] Aspergillus nidulans ATCC58396 and Aspergillus pachycristatus ATCC58397 are commonly used production strains for ECB production. The inventors found that the ECB synthesis gene clusters of the two strains contain two proteins AniJ (GenBank: AMM63165.1, strain ATCC58396) and EcdJ (GenBank: AFT91381.1, strain ATCC58397) with highly similar encoded proteins (sequence identity 84.63%) and unknown functions. The present invention found that overexpression of AniJ in ATCC58396 and overexpression of EcdJ in ATCC58397 can activate ECB biosynthesis genes and greatly increase the yield of ECB, proving that AniJ / EcdJ is a transcriptional activator of the ECB gene cluster. Based on this, two high-yield genetically engineered bacteria were obtained and applied to the efficient fermentation production of ECB.

[0005] As one aspect of the present invention, a genetically engineered bacterium with high yield of echinocandin B is provided, wherein the genetically engineered bacterium can overexpress the aniJ gene or the ecdJ gene.

[0006] The protein encoded by the aniJ gene is selected from:

[0007] (a) A protein consisting of the amino acid sequence in GenBank accession number AMM63165.1;

[0008] (b) a protein derived from the amino acid sequence of GenBank Accession No. AMM63165.1 by substitution, deletion and / or addition of one or more amino acid residues and having the same activity as GenBank Accession No. AMM63165.1;

[0009] or (c) other genes encoding proteins having an amino acid sequence composition that is more than 80% similar to that of GenBank Accession No. AMM63165.1;

[0010] The protein encoded by the ecdJ gene is selected from:

[0011] (a) A protein consisting of the amino acid sequence in GenBank accession number AFT91381.1;

[0012] (b) a protein derived from the amino acid sequence of GenBank Accession No. AFT91381.1 by substitution, deletion and / or addition of one or more amino acid residues and having the same activity as GenBank Accession No. AFT91381.1;

[0013] or (c) proteins encoded by other genes having an amino acid sequence composition with a similarity of more than 80% with that of GenBank Accession No. AFT91381.1.

[0014] Preferably, the starting bacteria of the genetically engineered bacteria is Aspergillus.

[0015] In an embodiment of the present invention, the Aspergillus is selected from Aspergillus nidulans ATCC58396 or Aspergillus pachycristatus ATCC58397.

[0016] As another aspect of the present invention, a method for constructing a genetically engineered bacterium is provided, wherein a homologous recombination method is used in Aspergillus nidulans ATCC58396, a glufosinate-ammonium resistance gene bar is used as a screening gene, and a strong promoter of Aspergillus nidulans is used to overexpress the gene aniJ in the echinocandin B biosynthesis gene cluster of the strain to obtain a recombinant bacterium.

[0017] In an embodiment of the present invention, a DNA sequence upstream of the start codon ATG of the aniJ gene is used as the homologous recombination fragment 1, and a DNA sequence downstream of the start codon ATG of the aniJ gene is used as the homologous recombination fragment 2; in a 5'-3' order, the homologous recombination fragment 1, the glufosinate resistance gene bar, the promoter Ptef1 and the homologous recombination fragment 2 are assembled into a DNA fragment as a transformation fragment by fusion PCR; this transformation fragment is transformed into Aspergillus nidulans ATCC58396 to obtain the engineered strain Aspergillus nidulans JOE.

[0018] The glufosinate resistance gene bar is shown in SEQ ID NO.1;

[0019] The promoter Ptef1 gene is shown in SEQ ID NO.2;

[0020] The homologous recombination fragment 1 gene is shown in SEQ ID NO.3;

[0021] The homologous recombination fragment 2 gene is shown in SEQ ID NO.4.

[0022] As another aspect of the present invention, a method for constructing a genetically engineered bacterium is provided, wherein a homologous recombination method is used in Aspergillus pachycristatus ATCC58397, a glufosinate-ammonium resistance gene bar is used as a screening gene, and a strong Aspergillus nidulans promoter is used to overexpress the gene ecdJ in the echinocandin B biosynthesis gene cluster of the Aspergillus pachycristatus ATCC58397 strain to obtain a recombinant bacterium.

[0023] In the embodiment of the present invention, a DNA sequence upstream of the start codon ATG of the ecdJ gene is used as homologous recombination fragment 3, and a segment downstream of the start codon ATG of the ecdJ gene is used as homologous recombination fragment 4; in a 5'-3' order, the homologous recombination fragment 3, the resistance gene bar, the promoter Ptef1 and the homologous recombination fragment 4 are assembled into a DNA fragment by fusion PCR as a transformation fragment. This transformation fragment is transformed into the Aspergillus pachycristatus ATCC58397 strain to obtain the engineered strain Aspergillus pachycristatus JOE;

[0024] The glufosinate resistance gene bar is shown in SEQ ID NO.1;

[0025] The promoter Ptef1 gene is shown in SEQ ID NO.2;

[0026] The homologous recombination fragment 3 gene is shown in SEQ ID NO.5;

[0027] The homologous recombination fragment 4 gene is shown in SEQ ID NO.6.

[0028] Specifically, the method for constructing the genetically engineered bacteria comprises the following steps:

[0029] Step 1, construction of aniJ or ecdJ overexpression gene elements;

[0030] Preferably, the amplified homologous recombination fragment 1 upstream of the start codon of the aniJ gene, the glufosinate resistance gene bar, the Ptef1 promoter and the homologous recombination fragment 2 downstream of the start codon of the aniJ gene are fused into a double-stranded DNA by fusion PCR, and the obtained DNA fragment is purified by gel recovery method;

[0031] Alternatively, the amplified homologous recombination fragment 3 upstream of the start codon of the ecdJ gene, the glufosinate resistance gene bar, the Ptef1 promoter and the homologous recombination fragment 4 downstream of the start codon of the ecdJ gene are fused into a double-stranded DNA by fusion PCR, and the obtained DNA fragments are purified by gel recovery method.

[0032] Step 2, protoplast transformation of aniJ overexpression gene elements:

[0033] After spore culture and germination of the starting strain, protoplasts were prepared;

[0034] Adding the DNA fragment obtained in step 1 to the protoplasts to obtain a mixture of DNA and cells for fungal transformation;

[0035] Then the positive colonies were screened by PCR.

[0036] Furthermore, the construction method also includes the cultivation and fermentation of genetically engineered bacteria.

[0037] As the third aspect of the present invention, it provides the use of the genetically engineered bacteria in the preparation of echinocandin B.

[0038] As a fourth aspect of the present invention, it is to provide the use of the encoded protein of aniJ or ecdJ gene as a transcriptional activator of the echinocandin B gene cluster, wherein the echinocandin B gene cluster includes the aniA-L gene in Aspergillus nidulans ATCC58396, and ecdA, ecdG-L, and htyA-E in Aspergillus pachycristatus ATCC58397.

[0039] The present invention also provides the use of overexpressing aniJ gene in Aspergillus nidulans ATCC58396 or overexpressing ecdJ gene in Aspergillus pachycristatus ATCC58397 in preparing echinocandin B, wherein the aniJ is shown in the sequence of GenBank accession number AMM63165.1, and the ecdJ is shown in the sequence of GenBank accession number AFT91381.1.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] 1. The present invention overexpresses aniJ in Aspergillus nidulans ATCC58396 and overexpresses ecdJ in Aspergillus pachycristatus ATCC58397. The yield of echinocandin B in the obtained genetically engineered bacteria is 10.2 times and 10.6 times that of wild bacteria, respectively, greatly improving the yield of ECB. Based on this, two high-yield genetically engineered bacteria are obtained and applied to the efficient fermentation production of ECB.

[0042] 2. The present invention also confirmed that overexpression of aniJ in Aspergillus nidulans ATCC58396 and overexpression of ecdJ in Aspergillus pachycristatus ATCC58397 can activate ECB biosynthesis genes, proving that the encoded proteins of aniJ and ecdJ genes are transcriptional activators of the ECB gene cluster, providing a new solution for the efficient production of ECB. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0044] Figure 1 Schematic diagram of the process of designing replacement of aniJ / ecdJ promoter for homologous recombination in Example 1 and Example 3;

[0045] Figure 2 The results of colony PCR of the engineering strains in Example 2 and Example 4 are as follows;

[0046] Figure 3 The expression level of the ani gene cluster after aniJ overexpression in Example 5 was determined;

[0047] Figure 4 Determination of the expression level of the ecd gene cluster after ecdJ overexpression in Example 5;

[0048] Figure 5 High-resolution mass spectrometry analysis of echinocandin B produced by the engineered strain in Example 6;

[0049] Figure 6 This is the metabolic profile of the wild bacteria and engineered bacteria in Example 6. DETAILED DESCRIPTION

[0050] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0051] Unless otherwise specified, the experimental methods used in the examples are conventional methods.

[0052] Unless otherwise specified, the materials and reagents used in the examples can be obtained from commercial sources.

[0053] Example 1, Construction of aniJ overexpression gene element

[0054] (1) Acquisition of relevant genes

[0055] Using the genome of Aspergillus nidulans ATCC58396 (ATCC, USA) as a template and SEQ ID NO.7 and SEQ ID NO.8 as primers, a homologous recombination fragment 1 of about 1000 bp upstream of the start codon of the aniJ gene was amplified, as shown in SEQ ID NO.3.

[0056] The glufosinate resistance gene bar was synthesized by GenScript Biotechnology Co., Ltd. The bar gene was amplified using the sequences SEQ ID NO.9 and SEQ ID NO.10 as primers, and the sequence was SEQ ID NO.1.

[0057] Using the genome of Aspergillus nidulans ATCC58396 (ATCC, USA) as a template and using sequences SEQ ID NO.11 and SEQ ID NO.12 as primers, the Ptef1 promoter sequence was amplified, as shown in SEQ ID NO.2.

[0058] Using the genome of Aspergillus nidulans ATCC58396 (ATCC, USA) as a template and using sequences SEQ ID NO.13 and SEQ ID NO.14 as primers, amplify the homologous recombination fragment 2 of about 1000 bp downstream of the start codon of the aniJ gene, as shown in SEQ ID NO.4.

[0059] (2) Construction of aniJ overexpression gene element

[0060] like Figure 1 As shown in A, the homologous recombination fragment 1, the glufosinate resistance gene bar, the Ptef1 promoter and the homologous recombination fragment 2 are fused into a double-stranded DNA by fusion PCR, and the obtained DNA fragment is purified by gel recovery method.

[0061] The polymerase used in the present invention is 2×Phanta Max Master Mix produced by Novagen. The 50μL amplification system is as follows: 1μL template, 1uL of upstream and downstream primers (10μM), 25μL of 2×Phanta Max Master Mix, and make up to 50μL with deionized water. The above PCR amplification conditions: 95℃, 5min; 95℃, 30S; 55℃, 30S; 72℃, 1min, 30 cycles; 72℃, 5min.

[0062] The 50 μL fusion PCR reaction system is as follows: 200 ng each of homologous recombination fragment 1, glufosinate resistance gene bar, Ptef1 promoter and homologous recombination fragment 2, 1 μL of front primer (SEQ ID NO.7), 1 μL of back primer (SEQ ID NO.14), 25 μL of 2×Phanta MaxMaster Mix, and fill to 50 μL with deionized water. Fusion PCR reaction conditions: 95°C, 5 min; 95°C, 30S; 55°C, 30S, 72°C, 4 min; 30 cycles; 72°C, 5 min.

[0063] The primer sequences for constructing the overexpression gene elements are shown in Table 1:

[0064] Table 1 Primer sequence list for constructing overexpression gene elements

[0065]

[0066]

[0067] Example 2, Protoplast transformation of aniJ overexpression gene element in Aspergillus nidulans ATCC58396

[0068] (1) Spore culture and germination

[0069] The spores of the starting strain Aspergillus nidulans ATCC58396 were spread on SMM solid medium (10 g / L glucose, 10 mM ammonium tartrate, 0.52 g / L KCl, 0.52 g / L MgSO4·7H2O, 1.52 g / L KH2PO4, 20 g / L agarose, 1 mL trace elements, pH = 6.5), and then placed in a 37°C incubator for inverted culture for 5 days until the spores grow. The spores were inoculated into 100 mL liquid SMM medium (10 g / L glucose, 10 mM ammonium tartrate, 0.52 g / L KCl, 0.52 g / L MgSO4·7H2O, 1.52 g / L KH2PO4, 1 mL trace elements, pH = 6.5), placed in a 37°C shaker, cultured for 12 hours at 220 rpm until the spores germinate. Take 50 mL of spore culture medium, centrifuge at 5000 rpm for 10 minutes at room temperature, remove the supernatant, and collect the germinated spores at the bottom of the centrifuge tube.

[0070] (2) Protoplast preparation

[0071] Take 15mg Lysing enzyme (Sigma-Aldrich, L3768-1G), 1g Vinotaste Pro (Novozymes) and 100mg snail enzyme (Sanggong Biology, A600870), dissolve them together in 30mL protoplast osmotic maintenance solution (1.2M magnesium sulfate, 6mM potassium dihydrogen phosphate, adjust pH to 5.8 with 1M potassium dihydrogen phosphate), and filter and sterilize with 0.22 micron sterile filter membrane to obtain enzymolysis solution. Weigh 0.5g of the germinated spores collected in step (1) of this embodiment, add them to 30mL enzymolysis solution, and enzymolysis for 6h in a shaking table at 28°C and 80rmp to remove the fungal cell wall. After the enzymolysis is completed, add three times the volume of STC buffer (1.2M sorbitol, 10mM CaCl2, 10mM Tris-HCl, pH 7.5. After high temperature sterilization, place at 4°C for storage), mix well and place in ice water for 15min. The mixture was then centrifuged at 5000 rpm and 4°C for 10 min, and the liquid on the centrifuge tube was removed. The protoplast cells at the bottom of the centrifuge tube were resuspended in 500 μL of STC buffer precooled in an ice water bath for subsequent transformation.

[0072] (3) Fungal transformation

[0073] Add 20 μg of DNA fragments obtained by gel recovery in step (2) of Example 1 to 100 μL of protoplasts obtained in step (2) of this Example, slowly blow 3-5 times with a pipette to mix, and place in an ice water bath for 1 hour. Then add 1 mL of PEG solution (60% PEG6000, 50 mM CaCl2, 10 mM Tris-HCl, pH 7.5, cooled to room temperature after high temperature sterilization) to the DNA and cell mixture, slowly blow 6-8 times with a pipette, and mix thoroughly. Then place in a 30°C incubator for 1 hour. Finally, the transformation mixture was spread on a screening medium (10 g / L glucose, 1.84 g / L ammonium tartrate, 0.52 g / L KCl, 0.52 g / LMgSO4·7H2O, 1.52 g / L KH2PO4, 218.6 g / L sorbitol, 4 g / L glufosinate, 20 g / L agarose, 1 mL trace elements, pH = 6.5) added with glufosinate and cultured for 3-4 days.

[0074] The trace element components in steps (1) and (3) are as follows (100 mL): 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, 5.00g Na4EDTA.

[0075] (4) PCR screening of positive clones

[0076] Take the fungal colonies grown in step (3) and use the fungal genome extraction kit (Solebo, D2300-100T) to extract genomic DNA. Using the above genomic DNA as a template, use primers SEQ ID NO.11 and SEQ ID NO.15 to perform colony PCR to verify whether the aniJ gene is replaced by the strong promoter Ptef1. The results are Figure 2 As shown, the theoretical PCR band is 1827 bp, which is consistent with the length of the colony PCR in the engineered strain, while no band was amplified in the wild strain Aspergillus nidulans ATCC58396. The positive colony was named Aspergillus nidulans JOE.

[0077] Example 3, Construction of ecdJ overexpression gene element

[0078] (1) Acquisition of relevant genes

[0079] Using the genome of Aspergillus pachycristatus ATCC58397 (ATCC, USA) as a template and sequences SEQ ID NO.16 and SEQ ID NO.17 as primers, a homologous recombination fragment 3 of about 1000 bp upstream of the start codon of the ecdJ gene was amplified, as shown in SEQ ID NO.5.

[0080] The glufosinate resistance gene bar was synthesized by GenScript Biotechnology Co., Ltd. The bar gene was amplified using the sequences SEQ ID NO.18 and SEQ ID NO.19 as primers, and the sequence was SEQ ID NO.1.

[0081] Using the genome of Aspergillus nidulans ATCC58396 as a template and sequences SEQ ID NO.20 and SEQ ID NO.21 as primers, the Ptef1 promoter sequence was amplified and is shown as SEQ ID NO.2.

[0082] Using the genome of Aspergillus pachycristatus ATCC58397 as a template and sequences SEQ ID NO.22 and SEQ ID NO.23 as primers, a homologous recombination fragment 4 of about 1000 bp downstream of the start codon of the ecdJ gene was amplified, as shown in SEQ ID NO.6.

[0083] (2) Construction of ecdJ overexpression gene element

[0084] like Figure 1 As shown in B, the homologous recombination fragment 3, the glufosinate resistance gene bar, the Ptef1 promoter and the homologous recombination fragment 4 are fused into a double-stranded DNA by fusion PCR, and the resulting DNA fragment is purified by gel recovery.

[0085] The polymerase used in the present invention is 2×Phanta Max Master Mix produced by Novagen. The 50μL amplification system is as follows: 1μL template, 1uL of upstream and downstream primers (10μM), 25μL of 2×Phanta Max Master Mix, and make up to 50μL with deionized water. The above PCR amplification conditions: 95℃, 5min; 95℃, 30S; 55℃, 30S; 72℃, 1min, 30 cycles; 72℃, 5min.

[0086] The 50 μL fusion PCR reaction system is as follows: 200 ng each of homologous recombination fragment 1, glufosinate resistance gene bar, Ptef1 promoter and homologous recombination fragment 2, 1 μL of front primer (SEQ ID NO.16), 1 μL of back primer (SEQ ID NO.23), 25 μL of 2×PhantaMax Master Mix, and fill to 50 μL with deionized water. Fusion PCR reaction conditions: 95°C, 5 min; 95°C, 30S; 55°C, 30S, 72°C, 4 min; 30 cycles; 72°C, 5 min.

[0087] Example 4, Protoplast transformation of ecdJ overexpression gene element in Aspergillus pachycristatus ATCC58397

[0088] (1) Spore culture and germination

[0089] The spores of the starting strain Aspergillus pachycristatus ATCC58397 were spread on SMM solid medium (10 g / L glucose, 10 mM ammonium tartrate, 0.52 g / L KCl, 0.52 g / L MgSO4·7H2O, 1.52 g / L KH2PO4, 20 g / L agarose, 1 mL trace elements, pH = 6.5), and then placed in a 37°C incubator for inverted culture for 5 days until the spores grow. The spores were inoculated into 100 mL liquid SMM medium (10 g / L glucose, 10 mM ammonium tartrate, 0.52 g / L KCl, 0.52 g / LMgSO4·7H2O, 1.52 g / L KH2PO4, 1 mL trace elements, pH = 6.5), placed in a 37°C shaker, cultured for 12 hours at 220 rpm until the spores germinate. Take 50 mL of spore culture medium, centrifuge at 5000 rpm for 10 minutes at room temperature, remove the supernatant, and collect the germinated spores at the bottom of the centrifuge tube.

[0090] (2) Protoplast preparation

[0091] Take 15 mg Lysing enzyme (Sigma-Aldrich, L3768-1G), 1 g Vinotaste Pro (Novozymes) and 100 mg snail enzyme (Sangon Biotechnology, A600870), dissolve them together in 30 mL protoplast osmotic maintenance solution (1.2 M magnesium sulfate, 6 mM potassium dihydrogen phosphate, pH adjusted to 5.8 with 1 M potassium dihydrogen phosphate), and filter and sterilize with a 0.22 μm sterile filter membrane to obtain an enzymatic solution. Weigh 0.5 g of the germinated spores collected in (1), add them to 30 mL of the enzymatic solution, and enzymolyze them in a shaking table at 28° C. and 80 rpm for 6 h to remove the fungal cell wall. After the enzymolysis is completed, add three times the volume of STC buffer (1.2 M sorbitol, 10 mM CaCl2, 10 mM Tris-HCl, pH 7.5. After high temperature sterilization, store at 4° C.), mix well, and place in ice water for 15 min. The mixture was then centrifuged at 5000 rpm and 4°C for 10 min, and the liquid on the centrifuge tube was removed. The protoplast cells at the bottom of the centrifuge tube were resuspended in 500 μL of STC buffer precooled in an ice water bath for subsequent transformation.

[0092] (3) Fungal transformation

[0093] Add 20 μg of DNA fragments obtained by gel recovery in step (2) of Example 3 to 100 μL of protoplasts obtained in step (2) of this Example, slowly blow 3-5 times with a pipette to mix, and place in an ice water bath for 1 hour. Then add 1 mL of PEG solution (60% PEG6000, 50 mM CaCl2, 10 mM Tris-HCl, pH 7.5. Cool to room temperature after high temperature sterilization) to the DNA and cell mixture, slowly blow 6-8 times with a pipette, and mix thoroughly. Then place in a 30°C incubator for 1 hour. Finally, the transformation mixture was spread on a screening medium (10 g / L glucose, 1.84 g / L ammonium tartrate, 0.52 g / L KCl, 0.52 g / LMgSO4·7H2O, 1.52 g / L KH2PO4, 218.6 g / L sorbitol, 4 g / L glufosinate, 20 g / L agarose, 1 mL trace elements, pH = 6.5) added with glufosinate and cultured for 3-4 days.

[0094] The trace element components in steps (1) and (3) are as follows (100 mL): 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, 5.00g Na4EDTA.

[0095] (4) PCR screening of positive clones

[0096] Take the fungal colonies grown in step (3) and use the fungal genome extraction kit (Solebo, D2300-100T) to extract genomic DNA. Using the above genomic DNA as a template, use primers SEQ ID NO.20 and SEQ ID NO.24 to perform colony PCR to verify whether the EcdJ gene is replaced by the strong promoter Ptef1. The results are Figure 2 As shown, the theoretical PCR band is 1828 bp, which is consistent with the length of the colony PCR in the engineered strain, while the wild strain Aspergillus pachycristatus ATCC 58397 did not amplify the band. The positive colony screened was named Aspergillus pachycristatus JOE.

[0097] Example 5 Determination of the expression level of the echinocandin B biosynthetic gene cluster in the engineered strain

[0098] (1) Cultivation and fermentation of strains

[0099] Spores of wild bacteria Aspergillus nidulans ATCC58396, Aspergillus pachycristatus ATCC58397, the engineered bacteria Aspergillus nidulans JOE prepared in Example 2, and Aspergillus pachycristatus JOE prepared in Example 4 were inoculated into 50 mL of liquid PDB seed culture medium (Solebo, P9240), respectively, and cultured at 28° C. and 220 rpm for 48 h. Then, 300 mL of fermentation medium (mannitol 100 g / L, glycerol 10 g / L, peptone 40 g / L, vegetable oil 10 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, pH 7.0) was placed in a 2 L shake flask and sterilized at high temperature, and 30 mL of seed culture solution was transferred to the shake flask and cultured in a shaker at 25°C and 220 rpm for 4-7 days.

[0100] (2) RNA extraction and RT-qPCR quantitative analysis of the echinocandin B gene cluster

[0101] Wild bacteria and engineered bacteria were cultured in fermentation medium for 4 days, and mycelia were collected by centrifugation. Mycelia were quickly frozen with liquid nitrogen and ground into powder in liquid nitrogen. Intracellular mRNA was extracted using an RNA extraction kit (Quick-RNA Fungal / BacterialMicroprep Kit (Zymo Research, R2010). Residual DNA was removed using DNase, and RNA was reverse transcribed using a reverse transcription kit RevertAid Reverse Transcriptase (Thermo Scientific EP0441).

[0102] RT-qPCR analysis was performed using a thermal cycler myiQ cycler (Bio-Rad). Take 1 μL of reverse transcription template, 1 μL of each front and back primer, 10 μL of ChamQ SYBR qPCR Master Mix (Vazyme, Q311-02), and 7 μL of deionized water and mix well as the reaction system. Three biological parallels were tested for each sample, and the β-actin gene (acn) AN6542 in the Aspergillus nidulans genome was used as an internal reference gene to correct the expression level. The test results are expressed as the ratio of gene expression in engineered bacteria to that in wild bacteria. After overexpression of gene aniJ, the expression results of the ani gene cluster are as follows: Figure 3 As shown in the figure: The expression levels of aniA-L genes in the echinocandin B biosynthesis gene cluster in the engineered strain Aspergillus nidulans JOE are higher than those in the wild strain Aspergillus nidulans ATCC58396. After the gene ecdJ is overexpressed, the expression level of the ecd gene cluster is as follows Figure 4 As shown: the expression levels of genes ecdA, ecdG-L, and htyA-E in the echinocandin B biosynthesis gene cluster in the engineered strain Aspergillus pachycristatus JOE were higher than those in the wild strain Aspergillus pachycristatus ATCC 58397. This result indicates that aniJ and ecdJ are transcriptional regulators of the echinocandin B gene cluster in Aspergillus nidulans ATCC58396 and Aspergillus pachycristatus ATCC58397, respectively.

[0103] The aniA-L gene, ecdA, ecdG-L and htyA-E related sequences analyzed by the RT-qPCR can be found in the NCBI database, and the gene accession number of NCBI is shown in Table 2. The primers used in RT-qPCR were designed with the assistance of primer-5 software.

[0104] Table 2 List of genes related to the biosynthetic gene cluster of echinocandin B in engineered strains

[0105]

[0106]

[0107] Example 6. Fermentation process of strains and qualitative and quantitative analysis of echinocandin B

[0108] (1) Extraction and purification of echinocandin B produced by strains

[0109] Take 1L of fermentation broth obtained in step (1) of Example 5 and centrifuge at 12000rmp and 4°C for 10min. Remove the supernatant, collect the bacterial cells, add 500mL of methanol and mix well, and place in a 4°C refrigerator for leaching for 2-3h. Then centrifuge at 12000rmp and 4°C for 10min, and transfer the upper methanol extract to a 2L beaker. Add 500mL of methanol to the lower bacterial cells again and repeat the above extraction steps to finally obtain 1L of methanol extract.

[0110] To prepare pure echinocandin B for high-resolution mass spectrometry analysis, 900 mL of the methanol extract obtained in step (2) was taken and evaporated to dryness using a rotary evaporator. The crude extract was dissolved with 10 mL of methanol and purified using a gel column (Sephadex LH-20). The product was collected by HPLC detection, and the gel-purified fraction containing echinocandin B was evaporated to dryness using a rotary evaporator, and then the evaporated sample was dissolved with methanol, and a semi-preparative column Ultimate XB-C18 column (10×250 mm, 5 μm, Yuexu Technology) was used for preparative liquid phase separation. The purified echinocandin B sample was subjected to high-resolution mass spectrometry analysis using Agilent 1290Infinity / 6230TOF LCMS, and the mass spectral data was detected using ESI positive ion mode.

[0111] Test results( Figure 5 ) showed that the molecular weights of the compounds produced by the engineered strains Aspergillus nidulans JOE and Aspergillus pachycristatus JOE were m / z 1082.5638 [M+Na] + and m / z 1082.5637[M+Na] + The molecular weight of the standard product of echinocandin B was m / z 1082.5637[M+Na] + These results are consistent with those of echinocandin B (C 52 H 81 N7O 16)Theoretical molecular weight m / z 1082.5637Da[M+Na] + Consistent.

[0112] (2) Quantitative analysis of echinocandin B produced by strains

[0113] The standard substance of Echinocandin B (Santa Cruz Biotechnology) was dissolved in methanol, and 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 500 mg / L, 800 mg / L, and 1000 mg / L standard solutions were prepared, and quantitative analysis was performed by HPLC. The corresponding peak area at 210 nm was read, and the concentration-peak area standard curve equation was obtained by linear fitting.

[0114] To determine the yield of echinocandin B produced by fungi, 0.5 mL of the methanol extract prepared in step (2) was taken and filtered through a 0.22 μm filter membrane for HPLC analysis. The echinocandin B produced by wild fungi Aspergillus nidulans ATCC58396, Aspergillus pachycristatus ATCC58397 and engineered fungi Aspergillus nidulans JOE and Aspergillus pachycristatus JOE was measured by HPLC and the corresponding peak areas were input into the standard curve equation to obtain the yields of the four fungi. The results are shown in the attached figure. Figure 6 As shown, overexpression of aniJ in Aspergillus nidulans ATCC58396 and overexpression of ecdJ in Aspergillus pachycristatus ATCC58397 can increase the production of echinocandin B.

[0115] The liquid chromatography conditions are as follows: the injection volume is 10 μL, the chromatographic column is a C18 column from Dalian Yilite Company, the specification is 5 μm, 250 mm×4.6 mm. The mobile phase A liquid is methanol, the mobile phase B liquid is water (containing 1‰ formic acid), the flow rate is 1 mL / min. The elution gradient is: 0-3 min, 5% A; 5-20 min, 5%-95% A; 20-25 min, 95% A, 25.1-30 min, 5% A. The UV detection wavelength is 210 nm, and the column temperature is 30°C.

[0116] The results of the strains' echinocandin B production showed that the echinocandin B production in Aspergillus nidulans JOE was 533.3 mg / L, which was 10.2 times that of the wild strain ATCC58396 (52.3 mg / L); the echinocandin B production in Aspergillus pachycristatus JOE was 625.7 mg / L, which was 10.6 times that of the wild strain ATCC58397 (58.6 mg / L).

[0117] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention, such as replacement of strong promoters, codon optimization of aniJ / ecdJ, homologous replacement of AniJ / EcdJ homologous proteins (similarity of more than 80%), should be included in the protection scope of the present invention.

Claims

1. A genetically engineered bacterium that produces high levels of echinocandin B, characterized in that: The genetically engineered bacteria overexpress the aniJ gene, and the protein encoded by the aniJ gene is a protein composed of the amino acid sequence in GenBank accession number AMM63165.1; The starting bacteria of the genetically engineered bacteria is Aspergillus; The Aspergillus is Aspergillus nidulans ATCC58396.

2. A method for constructing a genetically engineered bacterium, characterized in that: Homologous recombination was used in Aspergillus nidulans ATCC58396, the glufosinate resistance gene bar was used as a screening gene, and a strong Aspergillus nidulans promoter was used to overexpress the gene aniJ in the echinocandin B biosynthesis gene cluster of the Aspergillus nidulans ATCC58396 strain to obtain a recombinant bacterium.

3. The method for constructing a genetically engineered bacterium according to claim 2, characterized in that: A DNA sequence upstream of the start codon ATG of the aniJ gene is used as homologous recombination fragment 1, and a DNA sequence downstream of the start codon ATG of the aniJ gene is used as homologous recombination fragment 2; homologous recombination fragment 1, glufosinate resistance gene bar, promoter Ptef1 and homologous recombination fragment 2 are assembled into a DNA fragment as a transformation fragment by fusion PCR in a 5'-3' order; this transformation fragment is transformed into Aspergillus nidulans ATCC58396 to obtain an engineered strain Aspergillus nidulans JOE; The glufosinate resistance gene bar is shown in SEQ ID NO.1; The promoter Ptef1 gene is shown in SEQ ID NO.2; The homologous recombination fragment 1 gene is shown in SEQ ID NO.3; The homologous recombination fragment 2 gene is shown in SEQ ID NO.

4.

4. The method for constructing a genetically engineered bacterium according to claim 3, characterized in that: The steps include: Step 1, construction of aniJ overexpression gene element; The amplified homologous recombination fragment 1 upstream of the start codon of the aniJ gene, the glufosinate resistance gene bar, the Ptef1 promoter and the homologous recombination fragment 2 downstream of the start codon of the aniJ gene were fused into a double-stranded DNA by fusion PCR, and the obtained DNA fragments were purified by gel recovery method; Step 2, protoplast transformation of aniJ overexpression gene elements: After spore culture and germination of the starting strain, protoplasts were prepared; Adding the DNA fragment obtained in step 1 to the protoplasts to obtain a mixture of DNA and cells for fungal transformation; Then the positive colonies were screened by PCR.

5. Use of the genetically engineered bacterium according to claim 1 or the genetically engineered bacterium constructed by the construction method of any one of claims 2 to 4 in the preparation of echinocandin B.

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