CN117844657A - Genetically engineered bacterium for high-yield echinocandin B and application thereof - Google Patents

Abstract

The invention discloses a genetic engineering bacterium for high-yield echinocandin B and application thereof, wherein the genetic engineering bacterium overexpresses aniJ gene or ecdJ gene, and the starting bacterium of the genetic engineering bacterium is aspergillus. The invention also provides a construction method of the genetically engineered bacterium, which uses a homologous recombination method, uses glufosinate-ammonium resistance gene bar as a screening gene, and uses a strong aspergillus nidulans promoter to overexpress genes in a echinocandin B biosynthesis gene cluster of the strain to obtain recombinant bacterium. The yields of echinocandin B of the two genetically engineered bacteria are respectively 10.2 times and 10.6 times of that of the wild bacteria, and the yield of target products is greatly improved.

Description

Genetically engineered bacterium for high-yield echinocandin B and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and in particular relates to a genetic engineering bacterium for high-yield echinocandin B and application thereof.

Background

Anidulafungin is a lipopeptide antifungal drug of filamentous fungal origin, marketed by the company pyroxene in europe and the united states in 2007, with annual sales amounting to $ 1.33 billion (2019). The echinocandins can non-competitively inhibit beta-1, 3-glucose synthetase, prevent the synthesis of pathogenic fungi cell walls, and play a role in inhibiting the growth of fungi. In recent years, the market of antifungal drugs such as anidulafungin has been rapidly growing due to the rapid increase of cases of new and secondary invasive fungal infections caused by coronavirus infection, and the global market supply and demand is being met. However, the anidulafungin production process comprises two-step complex processes of fermentation production precursor echinocandin B (ECB) and chemical post-modification, and the clinical use price is high. At present, production strain optimization is still mainly based on mutation breeding. The production price of the bulk drug is high because of unstable fermentation yield of the mutagenized strain and numerous byproducts, and the large-scale production and clinical application of the anidulafungin are restricted.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a genetic engineering bacterium for high-yield echinocandin B and application thereof.

Aspergillus nidulans ATCC58396 and Aspergillus pachycristatus ATCC58397 are common production strains for ECB production. The inventors found that the ECB synthetic gene cluster of the two strains contained two proteins encoded highly similarly (sequence identity 84.63%), proteins of unknown function AniJ (GenBank: AMM63165.1, strain ATCC 58396) and EcdJ (GenBank: AFT91381.1, strain ATCC 58397). The invention discovers that the overexpression of AniJ in ATCC58396 and the overexpression of ecdJ in ATCC58397 can activate ECB biosynthesis genes, and can greatly improve the ECB yield, thereby proving that AniJ/ecdJ is a transcription activator of ECB gene clusters. Based on the method, two high-yield genetically engineered bacteria are obtained and are applied to ECB efficient fermentation production.

As one aspect of the present invention, there is provided a genetically engineered bacterium for high-yielding echinocandin B, which can overexpress the aniJ gene or overexpress the ecdJ gene,

the protein encoded by the aniJ gene is selected from:

(a) A protein consisting of the amino acid sequence in GenBank accession number AMM 63165.1;

(b) A derivative protein which is obtained by substituting and deleting and/or adding one or more amino acid residues of an amino acid sequence in GenBank accession number AMM63165.1 and has the same activity as that of GenBank accession number AMM 63165.1;

or (c) protein which is encoded by other genes and has more than 80 percent of similarity with the amino acid sequence composition shown in GenBank accession number AMM 63165.1;

the encoding protein of the ecdJ gene is selected from the group consisting of:

(a) A protein consisting of the amino acid sequence in GenBank accession number AFT 91381.1;

(b) A derivative protein which is obtained by substituting and deleting and/or adding one or more amino acid residues of an amino acid sequence in GenBank accession number AFT91381.1 and has the same activity as that of GenBank accession number AFT 91381.1;

or (c) protein which is encoded by other genes and has more than 80 percent of similarity with the amino acid sequence composition shown in the GenBank accession number AFT 91381.1.

Preferably, the starting strain of the genetically engineered strain is aspergillus.

In an embodiment of the invention, the aspergillus is selected from Aspergillus nidulans ATCC58396 or Aspergillus pachycristatus ATCC58397.

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

In the embodiment of the invention, a DNA sequence upstream of the ATG of the aniJ gene initiation codon is used as a homologous recombination fragment 1, and a DNA sequence downstream of the ATG of the aniJ gene initiation codon is used as a homologous recombination fragment 2; assembling the homologous recombination fragment 1, the glufosinate-ammonium resistance gene bar, the promoter Ptef1 and the homologous recombination fragment 2 into a DNA fragment serving as a transformation fragment in a 5'-3' sequence through fusion PCR; this transformed fragment was transformed into Aspergillus nidulans ATCC58396 to give engineering 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.

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

In the embodiment of the invention, a DNA sequence upstream of the ATG of the ecdJ gene initiation codon is used as a homologous recombination fragment 3, and a DNA sequence downstream of the ATG of the ecdJ gene initiation codon is used as a homologous recombination fragment 4; the homologous recombination fragment 3, the resistance gene bar, the promoter Ptef1 and the homologous recombination fragment 4 were assembled into a DNA fragment as a transformation fragment by means of fusion PCR in the 5'-3' order. Transforming the transformed fragment into Aspergillus pachycristatus ATCC58397 strain to obtain engineering strain Aspergillus pachycristatus 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 3 gene is shown in SEQ ID NO. 5;

the homologous recombination fragment 4 gene is shown in SEQ ID NO. 6.

Specifically, the construction method of the genetically engineered bacterium comprises the following steps:

step 1, constructing an aniJ or ecdJ over-expression gene element;

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

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

Step 2 protoplast transformation of anij over-expressed gene element:

preparing protoplast after spore culture and germination of the original strain;

adding the DNA fragment obtained in the step 1 into protoplast to obtain a mixture of DNA and cells, and performing fungal transformation;

positive colonies were then screened by PCR.

Furthermore, the construction method also comprises the culture and fermentation of the genetically engineered bacteria.

As a third aspect of the invention, there is provided an application of the genetically engineered bacterium in preparation of echinocandin B.

As a fourth aspect of the present invention, there is provided the use of a protein encoding an aniJ or ecdJ gene as a transcriptional activator of an echinocandin B gene cluster comprising the aniA-L gene in Aspergillus nidulans ATCC58396 and ecdA, ecdG-L, htyA-E in Aspergillus pachycristatus ATCC58397.

The invention also provides the use of an aniJ gene as shown in the sequence in GenBank accession number AMM63165.1 or an ecdJ gene as shown in the sequence in GenBank accession number AFT91381.1 in Aspergillus nidulans ATCC58396 or Aspergillus pachycristatus ATCC58397 for the preparation of echinocandin B.

Compared with the prior art, the invention has the beneficial effects that:

1, the invention over-expresses aniJ in Aspergillus nidulans ATCC58396 and ecdJ in Aspergillus pachycristatus ATCC58397, and the yield of echinocandin B of the obtained genetically engineered bacterium is 10.2 times and 10.6 times of that of wild bacterium respectively, thus greatly improving the yield of ECB. Based on the method, two high-yield genetically engineered bacteria are obtained and are applied to ECB efficient fermentation production.

2, the invention also proves that the ECB biosynthesis genes can be activated by over-expressing aniJ in Aspergillus nidulans ATCC58396 and by over-expressing ecdJ in Aspergillus pachycristatus ATCC58397, and the coded proteins of aniJ and ecdJ genes are proved to be transcription activating factors of ECB gene clusters, thus providing a new scheme for the efficient production of ECB.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

FIG. 1 is a schematic diagram showing the process of substituting aniJ/ecdJ promoters by homologous recombination design in example 1 and example 3;

FIG. 2 shows PCR results for colonies of engineering strains in example 2 and example 4;

FIG. 3 is a graph showing measurement of the expression level of an ani gene cluster after aniJ overexpression in example 5;

FIG. 4 is a graph showing the measurement of the expression level of ecd gene cluster after overexpression of ecdJ in example 5;

FIG. 5 is a high resolution mass spectrometry analysis of echinocandin B produced by the engineering strain of example 6;

FIG. 6 is a metabolic profile of wild-type bacteria and engineered bacteria of example 6.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The experimental methods used in the examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples are commercially available unless otherwise specified.

EXAMPLE 1 construction of an aniJ overexpressing Gene element

(1) Acquisition of related genes

The Aspergillus nidulans ATCC58396 (ATCC) genome is used as a template, the sequences SEQ ID NO.7 and SEQ ID NO.8 are used as primers, and the homologous recombination fragment 1 with about 1000bp upstream of the initiation codon of the aniJ gene is obtained by amplification, and is shown as SEQ ID NO. 3.

The glufosinate-ammonium resistance gene bar gold sry biotechnology limited company. The bar gene is obtained by amplification with the sequences SEQ ID NO.9 and SEQ ID NO.10 as primers, and the sequence is SEQ ID NO.1.

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

A homologous recombination fragment 2 of about 1000bp downstream of the initiation codon of the aniJ gene was amplified using the Aspergillus nidulans ATCC58396 (American, ATCC) genome as a template and the sequences SEQ ID NO.13 and SEQ ID NO.14 as primers, as shown in SEQ ID NO. 4.

(2) Construction of an aniJ overexpression Gene element

As shown in FIG. 1A, the homologous recombination fragment 1, the glufosinate resistance gene bar, the Ptef1 promoter and the homologous recombination fragment 2 were fused into a double-stranded DNA by fusion PCR, and the resulting DNA fragment was purified by a gel recovery method.

The polymerase used in the present invention was 2X Phanta Max Master Mix produced by Northenozan. The 50. Mu.L amplification system was as follows: 1. Mu.L of template, 1. Mu.L of upstream and downstream primers (10. Mu.M) each, 2X Phanta Max Master Mix. Mu.L, was filled to 50. Mu.L with deionized water. The PCR amplification conditions described above: 95 ℃ for 5min;95 ℃ and 30S;55 ℃,30S;72 ℃,1min,30 cycles; 72℃for 5min.

The 50. Mu.L fusion PCR reaction system was as follows: 200ng each of homologous recombination fragment 1, glufosinate resistance gene bar, ptef1 promoter and homologous recombination fragment 2, 1. Mu.L of the front primer (SEQ ID NO. 7), 1. Mu.L of the rear primer (SEQ ID NO. 14), 2X Phanta Max Master Mix. Mu.L, and 50. Mu.L of the rear primer were made up with deionized water. Fusion PCR reaction conditions: 95 ℃ for 5min;95 ℃ and 30S;55 ℃,30S,72 ℃ for 4min;30 cycles; 72℃for 5min.

The sequences of the primers used to construct the over-expressed gene elements are shown in Table 1:

TABLE 1 construction primer sequence Listing of over-expressed Gene elements

Protoplast transformation of an aniJ overexpressing Gene element in example 2,Aspergillus nidulans ATCC58396

(1) Spore culture and germination

Spores of the starting strain Aspergillus nidulans ATCC58396 were plated on SMM solid medium (glucose 10g/L,10mM ammonium tartrate, 0.52g/L KCl,0.52g/L MgSO) ₄ ·7H ₂ O，1.52g/L KH ₂ PO ₄ 20g/L agarose, 1mL trace element, pH=6.5) and then placed in an incubator at 37℃for 5 days in an inverted culture until spores grow out. Spores were inoculated into 100mL of liquid SMM medium (glucose 10g/L,10mM ammonium tartrate, 0.52g/L KCl,0.52g/L MgSO) ₄ ·7H ₂ O，1.52g/L KH ₂ PO ₄ 1mL of trace element, ph=6.5), placed on a shaker at 37 ℃ and incubated for 12 hours at 220rmp until spores germinate. Taking 50mL of spore culture medium, centrifuging at 5000rmp for 10 minutes at room temperature, removing the supernatant, and collecting a centrifuge tubeSpores germinate at the bottom.

(2) Protoplast preparation

15mg of Lysing enzyme (Sigma-Aldrich, L3768-1G), 1G of Vinostaste Pro (Norwestine) and 100mg of snailase (Bio, A600870) were dissolved together in 30mL of protoplast penetration maintenance solution (1.2M magnesium sulfate, 6mM dipotassium hydrogen phosphate, pH adjusted to 5.8 with 1M potassium dihydrogen phosphate) and sterilized by filtration through a 0.22 μm sterile filter to obtain an enzymatic hydrolysate. 0.5g of the germinated spores collected in step (1) of this example were weighed, added to 30mL of the enzymatic hydrolysate, and subjected to enzymatic hydrolysis in an 80rmp shaker at 28℃for 6 hours to remove the fungal cell walls. After the completion of the enzymatic hydrolysis, three volumes of STC buffer (1.2M sorbitol, 10mM CaCl) were added to the enzymatic hydrolysate ₂ 10mM Tris-HCl, pH 7.5. Sterilizing at high temperature, storing at 4deg.C), mixing, and standing in ice water for 15min. The mixture was centrifuged at 5000rmp at 4℃for 10min, and the liquid above the centrifuge tube was removed. Protoplast cells at the bottom of the centrifuge tube were resuspended in 500 μl of ice-water bath pre-chilled STC buffer for subsequent transformation.

(3) Fungal transformation

20. Mu.g of the DNA fragment recovered in step (2) of example 1 was added to 100. Mu.L of the protoplast obtained in step (2) of this example, and the mixture was slowly flushed 3-5 times with a pipette, and placed in an ice-water bath for 1 hour. 1mL of PEG solution (60% PEG6000, 50mM CaCl) was then added to the DNA and cell mixture ₂ 10mM Tris-HCl, pH 7.5, sterilized at high temperature and cooled to room temperature), slowly purged 6-8 times with a pipette, and thoroughly mixed. Then, the mixture was placed in an incubator at 30℃for 1 hour. Finally, the transformation mixture was spread on a screening medium (glucose 10g/L,1.84g/L ammonium tartrate, 0.52g/L KCl,0.52 g/LMgSO) ₄ ·7H ₂ O，1.52g/L KH ₂ PO ₄ 218.6g/L sorbitol, 4g/L glufosinate, 20g/L agarose, 1mL trace element, pH=6.5) for 3-4 days.

The trace element components in the steps (1) and (3) are as follows (100 mL): 2.20g ZnSO ₄ ·7H ₂ O，1.10g H ₃ BO ₃ ，0.50g MnCl ₂ ·4H ₂ O，0.16g FeSO ₄ ·7H ₂ O，0.16g CoCl ₂ ·5H ₂ O，0.16g CuSO ₄ ·5H ₂ O，0.11g(NH ₄ ) ₆ Mo ₇ O ₂₄ ·4H ₂ O，5.00g Na ₄ EDTA。

(4) PCR screening of Positive clones

Taking fungus colonies growing in the step (3), and respectively extracting genome DNA by using a fungus genome extraction kit (Soy pal, D2300-100T). The above genomic DNA was used as a template, and colony PCR was performed using primers SEQ ID NO.11 and SEQ ID NO.15 to verify whether the aniJ gene was replaced with a strong promoter Ptef1. As a result, as shown in FIG. 2, the theoretical PCR band was 1827bp consistent with the colony PCR length in the engineering strain, whereas the wild strain Aspergillus nidulans ATCC58396 did not amplify the band. The positive colony was designated Aspergillus nidulans JOE.

Example 3 construction of ecdJ overexpression Gene element

(1) Acquisition of related genes

The Aspergillus pachycristatus ATCC58397 (ATCC) genome is used as a template, the sequences SEQ ID NO.16 and SEQ ID NO.17 are used as primers, and the homologous recombination fragment 3 of about 1000bp upstream of the ecdJ gene start codon is obtained by amplification, and is shown as SEQ ID NO. 5.

The glufosinate-ammonium resistance gene bar gold sry biotechnology limited company. The bar gene is amplified by using the sequences SEQ ID NO.18 and SEQ ID NO.19 as primers, and the sequence is SEQ ID NO.1.

The Aspergillus nidulans ATCC58396 genome is used as a template, the sequences SEQ ID NO.20 and SEQ ID NO.21 are used as primers, and a Ptef1 promoter sequence is obtained by amplification and is shown as SEQ ID NO. 2.

The Aspergillus pachycristatus ATCC58397 genome is used as a template, the sequences SEQ ID NO.22 and SEQ ID NO.23 are used as primers, and the homologous recombination fragment 4 of about 1000bp downstream of the ecdJ gene start codon is obtained through amplification, and the homologous recombination fragment is shown in SEQ ID NO. 6.

(2) Construction of ecdJ overexpression Gene elements

As shown in FIG. 1B, the homologous recombination fragment 3, the glufosinate resistance gene bar, the Ptef1 promoter and the homologous recombination fragment 4 were fused into a double-stranded DNA by fusion PCR, and the resulting DNA fragment was purified by a gel recovery method.

The polymerase used in the present invention was 2X Phanta Max Master Mix produced by Northenozan. The 50. Mu.L amplification system was as follows: 1. Mu.L of template, 1. Mu.L of upstream and downstream primers (10. Mu.M) each, 2X Phanta Max Master Mix. Mu.L, was filled to 50. Mu.L with deionized water. The PCR amplification conditions described above: 95 ℃ for 5min;95 ℃ and 30S;55 ℃,30S;72 ℃,1min,30 cycles; 72℃for 5min.

The 50. Mu.L fusion PCR reaction system was as follows: 200ng each of homologous recombination fragment 1, glufosinate resistance gene bar, ptef1 promoter and homologous recombination fragment 2, 1. Mu.L of the front primer (SEQ ID NO. 16), 1. Mu.L of the rear primer (SEQ ID NO. 23), 2X Phanta Max Master Mix. Mu.L, and 50. Mu.L of the rear primer (SEQ ID NO. 23) were filled with deionized water. Fusion PCR reaction conditions: 95 ℃ for 5min;95 ℃ and 30S;55 ℃,30S,72 ℃ for 4min;30 cycles; 72℃for 5min.

Protoplast transformation of ecdJ overexpressing Gene elements in example 4,Aspergillus pachycristatus ATCC58397

(1) Spore culture and germination

Spores of the starting strain Aspergillus pachycristatus ATCC58397 were plated on SMM solid medium (glucose 10g/L,10mM ammonium tartrate, 0.52g/L KCl,0.52g/L MgSO) ₄ ·7H ₂ O，1.52g/L KH ₂ PO ₄ 20g/L agarose, 1mL trace element, pH=6.5) and then placed in an incubator at 37℃for 5 days in an inverted culture until spores grow out. Spores were inoculated into 100mL of liquid SMM medium (glucose 10g/L,10mM ammonium tartrate, 0.52g/L KCl,0.52g/L MgSO) ₄ ·7H ₂ O，1.52g/L KH ₂ PO ₄ 1mL of trace element, ph=6.5), placed on a shaker at 37 ℃ and incubated for 12 hours at 220rmp until spores germinate. 50mL of spore culture medium is taken, and is centrifuged for 10 minutes at 5000rmp at room temperature, the supernatant is removed, and germinated spores at the bottom of the centrifuge tube are collected.

(2) Protoplast preparation

15mg of Lysing enzyme (Sigma-Aldrich, L3768-1G), 1G of Vinostaste Pro (Norwestine) and 100mg of snailase (Bio, A600870) were dissolved together in 30mL of protoplast penetration maintenance solution (1.2M magnesium sulfate, 6mM dipotassium hydrogen phosphate, 1M phosphate)Potassium dihydrogen is adjusted to pH 5.8), and the enzymolysis liquid is obtained by filtering and sterilizing with a 0.22 micron sterile filter membrane. 0.5g of the germinated spores collected in (1) is weighed, added into 30mL of enzymolysis liquid, and subjected to enzymolysis for 6 hours at 28 ℃ in an 80rmp shaking table, and fungus cell walls are removed. After the completion of the enzymatic hydrolysis, three volumes of STC buffer (1.2M sorbitol, 10mM CaCl) were added to the enzymatic hydrolysate ₂ 10mM Tris-HCl, pH 7.5. Sterilizing at high temperature, storing at 4deg.C), mixing, and standing in ice water for 15min. The mixture was centrifuged at 5000rmp at 4℃for 10min, and the liquid above the centrifuge tube was removed. Protoplast cells at the bottom of the centrifuge tube were resuspended in 500 μl of ice-water bath pre-chilled STC buffer for subsequent transformation.

(3) Fungal transformation

20. Mu.g of the DNA fragment recovered in step (2) of example 3 was added to 100. Mu.L of the protoplast obtained in step (2) of this example, and the mixture was slowly flushed 3-5 times with a pipette, and placed in an ice-water bath for 1 hour. 1mL of PEG solution (60% PEG6000, 50mM CaCl) was then added to the DNA and cell mixture ₂ 10mM Tris-HCl, pH 7.5. Cooling to room temperature after high temperature sterilization), slowly purging with a pipette for 6-8 times, and fully mixing. Then, the mixture was placed in an incubator at 30℃for 1 hour. Finally, the transformation mixture was spread on a screening medium (glucose 10g/L,1.84g/L ammonium tartrate, 0.52g/L KCl,0.52 g/LMgSO) ₄ ·7H ₂ O，1.52g/L KH ₂ PO ₄ 218.6g/L sorbitol, 4g/L glufosinate, 20g/L agarose, 1mL trace element, pH=6.5) for 3-4 days.

The trace element components in the steps (1) and (3) are as follows (100 mL): 2.20g ZnSO ₄ ·7H ₂ O，1.10g H ₃ BO ₃ ，0.50g MnCl ₂ ·4H ₂ O，0.16g FeSO ₄ ·7H ₂ O，0.16g CoCl ₂ ·5H ₂ O，0.16g CuSO ₄ ·5H ₂ O，0.11g(NH ₄ ) ₆ Mo ₇ O ₂₄ ·4H ₂ O，5.00g Na ₄ EDTA。

(4) PCR screening of Positive clones

Taking fungus colonies growing in the step (3), and respectively extracting genome DNA by using a fungus genome extraction kit (Soy pal, D2300-100T). The above genomic DNA was used as a template, and colony PCR was performed using primers SEQ ID NO.20 and SEQ ID NO.24 to verify whether the ecdJ gene was replaced with a strong promoter Ptef1. As a result, as shown in FIG. 2, the theoretical PCR band was 1828bp consistent with the colony PCR length in the engineering strain, whereas the wild strain Aspergillus pachycristatus ATCC58397 did not amplify the band. The positive colonies screened were designated Aspergillus pachycristatus JOE.

Example 5 determination of expression level of echinocandin B biosynthetic Gene Cluster of engineering Strain

(1) Cultivation and fermentation of strains

Spores of wild fungi Aspergillus nidulans ATCC58396 and Aspergillus pachycristatus ATCC58397, engineering bacteria Aspergillus nidulans JOE prepared in example 2 and Aspergillus pachycristatus JOE prepared in example 4 were inoculated into 50mL of liquid PDB seed medium (Soxhibao, P9240) and cultured at 28℃and 220rmp for 48 hours. Then 300mL of fermentation medium (mannitol 100g/L, glycerol 10g/L, peptone 40g/L, vegetable oil 10g/L, K) ₂ HPO ₄ ·3H ₂ O8g/L，MgSO ₄ ·7H ₂ O 0.5g/L，MnSO ₄ ·H ₂ O 0.2g/L，FeSO ₄ ·7H ₂ O 0.05g/L，CaCl ₂ 0.5g/L，CuSO ₄ ·5H ₂ O0.6 g/L, pH 7.0) was sterilized in 2L shaking flask at high temperature, 30mL of seed culture solution was transferred to the shaking flask, and cultured in 220rmp shaking table at 25℃for 4-7 days.

(2) Extraction of RNA and quantitative RT-qPCR analysis of echinocandin B Gene Cluster

Culturing wild fungus and engineering fungus in fermentation medium for 4 days, and centrifuging to collect mycelium. The mycelia were quickly flash frozen with liquid nitrogen, and the mycelia were ground to powder in liquid nitrogen, intracellular mRNA was extracted using RNA extraction kit (Quick-RNA Fungal/Bacterial Microprep Kit (Zymo Research, R2010)) using DNase to remove residual DNA and reverse transcribed using reverse transcription kit RevertAid Reverse Transcriptase (Thermo Scientific EP 0441).

RT-qPCR analysis was performed using a thermocycler myiQ cycler (Bio-Rad). mu.L of reverse transcription template, 1. Mu.L of each of the front and rear primers, 10. Mu. L ChamQ SYBR qPCR Master Mix (Vazyme, Q311-02) and 7. Mu.L of deionized water were mixed uniformly to prepare a reaction system. Three biologically parallel, aspergillus nidulans genomes were tested for beta-actin gene (acn) AN6542 as AN internal reference gene for correction of expression levels per sample. The detection result is expressed by the ratio of the gene expression quantity in engineering bacteria to the expression quantity of wild bacteria. After the gene aniJ is over-expressed, the expression quantity result of the ani gene cluster is shown in FIG. 3: the expression level of the gene aniA-L in the echinocandin B biosynthesis gene cluster in the engineering bacterium Aspergillus nidulans JOE is higher than that of the wild strain Aspergillus nidulans ATCC58396. After the gene ecdJ is over-expressed, the expression quantity result of the ecd gene cluster is shown in fig. 4: the expression level of the gene ecdA, ecdG-L and htyA-E in the echinocandin B biosynthesis gene cluster in the engineering bacterium Aspergillus pachycristatus JOE is higher than that of the wild strain Aspergillus pachycristatus ATCC58397. 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.

The related sequences of aniA-L gene, ecdA, ecdG-L and htyA-E analyzed by RT-qPCR can be found in NCBI database, and the gene search number of NCBI is shown in Table 2. Primers used for RT-qPCR were designed with the aid of primer-5 software.

TABLE 2 echinocandin B biosynthetic gene cluster related Gene List of engineering Strain

Example 6 fermentation of the Strain and qualitative and quantitative analysis of echinocandin B

(1) Leaching and purifying echinocandin B produced by bacterial strain

1L of the fermentation broth obtained in the step (1) of example 5 was centrifuged at 12000rmp at 4℃for 10min. Removing the supernatant, collecting thallus, adding 500mL of methanol, mixing, and leaching in a refrigerator at 4deg.C for 2-3 hr. The upper methanol extract was then transferred to a 2L beaker by centrifugation at 12000rmp at 4℃for 10min. 500mL of methanol was added again to the lower cell, and the above leaching step was repeated, to finally obtain 1L of methanol leaching solution.

To prepare pure echinocandin B for high resolution mass spectrometry, 900mL of the methanol extract obtained in step (2) was taken and evaporated to dryness using a rotary evaporator. The crude extract was dissolved with 10mL of methanol and purified by 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, then the evaporated sample was dissolved with methanol and used for preparative liquid phase separation using semi-preparative column Ultimate XB-C18 column (10X 250mm,5 μm, yuehu technology). The purified echinocandin B samples were subjected to high resolution mass spectrometry using Agilent 1290Infinity/6230TOF LCMS and mass spectrometry data were detected using ESI positive ion mode.

The results of the test (FIG. 5) show that the molecular weights of the compounds produced by the engineering strains Aspergillus nidulans JOE and Aspergillus pachycristatus JOE are m/z 1082.5638[ M+Na ], respectively] ⁺ And m/z 1082.5637[ M+Na ]] ⁺ Echinocandin B standard with a measured molecular weight of m/z 1082.5637[ M+Na ]] ⁺ . These results are all similar to those of echinocandin B (C ₅₂ H ₈₁ N ₇ O ₁₆ ) Theoretical molecular weight m/z 1082.5637Da [ M+Na ]] ⁺ And consistent.

(2) Quantitative analysis of echinocandin B produced by the Strain

Echinocandin B standard (Santa Cruz Biotechnology) was dissolved in methanol to prepare 50mg/L,100mg/L,200mg/L,300mg/L,500mg/L,800mg/L,1000mg/L standard solutions, respectively, and quantitatively analyzed by HPLC. The corresponding peak area at 210nm was read and a concentration-peak area standard curve equation was obtained using linear fitting.

To determine the yield of echinocandin B produced by the fungus, 0.5mL of the methanol extract prepared in step (2) was taken and filtered through a 0.22 μm filter for HPLC analysis. And (3) inputting peak areas corresponding to the echinocandin B produced by the wild bacteria Aspergillus nidulans ATCC58396 and Aspergillus pachycristatus ATCC58397 and the engineering bacteria Aspergillus nidulans JOE and Aspergillus pachycristatus JOE after HPLC measurement into a standard curve equation to obtain the yield of four strains. As a result, as shown in FIG. 6, overexpression of aniJ in strain Aspergillus nidulans ATCC58396 and ecdJ in strain Aspergillus pachycristatus ATCC58397 both increased the production of echinocandin B.

The liquid chromatography conditions: the sample amount was 10. Mu.L, and the column was a C18 column of Lian Yili Tex, 5 μm,250 mm. Times.4.6 mm. The mobile phase A liquid is methanol, the liquid B is water (containing 1%o formic acid), and the flow rate is as follows: 1mL/min. Elution gradient: 0-3min,5% A;5-20min,5% -95% A;20-25min,95% A,25.1-30min,5% A. The UV detection wavelength was 210nm and the column temperature was 30deg.C.

The result of the yield of the echinocandin B of the strain shows that the yield of the echinocandin B is 533.3mg/L at Aspergillus nidulans JOE, which is 10.2 times that of the strain of the wild fungus ATCC58396 (52.3 mg/L); the yield of echinocandin B was 625.7mg/L at Aspergillus pachycristatus JOE, 10.6 times the yield of wild type ATCC58397 strain (58.6 mg/L).

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent substitution, improvement, etc., of a strong promoter, codon optimization of aniJ/ecdJ, and homologous substitution of an aniJ/ecdJ homologous protein (similarity of 80% or more) are included in the scope of the present invention.

Claims (10)

1. The genetically engineered bacterium for high-yield echinocandin B is characterized in that the genetically engineered bacterium overexpresses aniJ gene or ecdJ gene,

the protein encoded by the aniJ gene is selected from:

(a) A protein consisting of the amino acid sequence in GenBank accession number AMM 63165.1;

(b) A derivative protein which is obtained by substituting and deleting and/or adding one or more amino acid residues of an amino acid sequence in GenBank accession number AMM63165.1 and has the same activity as that of GenBank accession number AMM 63165.1;

or (c) protein which is encoded by other genes and has more than 80 percent of similarity with the amino acid sequence composition shown in GenBank accession number AMM 63165.1;

the encoding protein of the ecdJ gene is selected from the group consisting of:

(a) A protein consisting of the amino acid sequence in GenBank accession number AFT 91381.1;

(b) A derivative protein which is obtained by substituting and deleting and/or adding one or more amino acid residues of an amino acid sequence in GenBank accession number AFT91381.1 and has the same activity as that of GenBank accession number AFT 91381.1;

or (c) protein which is encoded by other genes and has more than 80 percent of similarity with the amino acid sequence composition shown in the GenBank accession number AFT 91381.1.

2. The genetically engineered bacterium for high yield of echinocandin B of claim 1, wherein the starting bacterium of the genetically engineered bacterium is aspergillus.

3. The genetically engineered bacterium for high yield of echinocandin B of claim 2, wherein the aspergillus is selected from Aspergillus nidulans ATCC58396 or Aspergillus pachycristatus ATCC58397.

4. A construction method of genetic engineering bacteria is characterized in that a homologous recombination method is utilized in Aspergillus nidulans ATCC58396, glufosinate-ammonium resistance gene bar is used as a screening gene, and a strong aspergillus nidulans promoter is used for over-expression of a gene aniJ in a Aspergillus nidulans ATCC58396 strain echinocandin B biosynthesis gene cluster to obtain recombinant bacteria.

5. The method for constructing a genetically engineered bacterium according to claim 4, wherein a DNA sequence upstream of the ATG of the aniJ gene start codon is used as the homologous recombination fragment 1, and a DNA sequence downstream of the ATG of the aniJ gene start codon is used as the homologous recombination fragment 2; assembling the homologous recombination fragment 1, the glufosinate-ammonium resistance gene bar, the promoter Ptef1 and the homologous recombination fragment 2 into a DNA fragment serving as a transformation fragment in a 5'-3' sequence through fusion PCR; transforming the transformed fragment into Aspergillus nidulans ATCC58396 to obtain an engineering 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.

6. A construction method of genetic engineering bacteria is characterized in that a homologous recombination method is utilized in Aspergillus pachycristatus ATCC58397, glufosinate-ammonium resistance gene bar is used as a screening gene, and a strong promoter of aspergillus nidulans is used for over-expression of gene ecdJ in Aspergillus pachycristatus ATCC58397 strain echinocandin B biosynthesis gene cluster to obtain recombinant bacteria.

7. The method according to claim 6, wherein a DNA sequence upstream of ATG of the ecdJ gene is used as the homologous recombination fragment 3, and a DNA sequence downstream of ATG of the ecdJ gene is used as the homologous recombination fragment 4; assembling the homologous recombination fragment 3, the glufosinate-ammonium resistance gene bar, the promoter Ptef1 and the homologous recombination fragment 4 into a DNA fragment serving as a transformation fragment in a 5'-3' sequence through fusion PCR; transforming the transformed fragment into Aspergillus pachycristatus ATCC58397 strain to obtain engineering strain Aspergillus pachycristatus 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 3 gene is shown in SEQ ID NO. 5;

the homologous recombination fragment 4 gene is shown in SEQ ID NO. 6.

8. The method for constructing a genetically engineered bacterium according to any one of claims 4 to 7, comprising the steps of:

step 1, constructing an aniJ or ecdJ over-expression gene element;

fusing the amplified homologous recombination fragment 1 upstream of the initial codon of the aniJ gene, the glufosinate-ammonium resistance gene bar, the Ptef1 promoter and the homologous recombination fragment 2 downstream of the initial codon of the aniJ gene into double-stranded DNA through fusion PCR, and purifying the obtained DNA fragment by a glue recovery method;

or fusing the amplified homologous recombination fragment 3 at the upstream of the ecdJ gene start codon, the glufosinate-ammonium resistance gene bar, the Ptef1 promoter and the homologous recombination fragment 4 at the downstream of the ecdJ gene start codon into a double-stranded DNA through fusion PCR, and purifying the obtained DNA fragment by a gel recovery method;

step 2 protoplast transformation of anij over-expressed gene element:

preparing protoplast after spore culture and germination of the original strain;

adding the DNA fragment obtained in the step 1 into protoplast to obtain a mixture of DNA and cells, and performing fungal transformation;

positive colonies were then screened by PCR.

9. The use of the genetically engineered bacterium according to any one of claims 1 to 8 in the preparation of echinocandin B.

The use of the protein encoded by the aniJ or ecdJ gene as a transcriptional activator for the echinocandin B gene cluster,

the protein encoded by the aniJ gene is selected from:

(a) A protein consisting of the amino acid sequence in GenBank accession number AMM 63165.1;

(b) A derivative protein which is obtained by substituting and deleting and/or adding one or more amino acid residues of an amino acid sequence in GenBank accession number AMM63165.1 and has the same activity as that of GenBank accession number AMM 63165.1;

or (c) protein which is encoded by other genes and has more than 80 percent of similarity with the amino acid sequence composition shown in GenBank accession number AMM 63165.1;

the encoding protein of ecdJ gene is selected from:

(a) A protein consisting of the amino acid sequence in GenBank accession number AFT 91381.1;

(b) A derivative protein which is obtained by substituting and deleting and/or adding one or more amino acid residues of an amino acid sequence in GenBank accession number AFT91381.1 and has the same activity as that of GenBank accession number AFT 91381.1;

or (c) protein which is encoded by other genes and has more than 80 percent of similarity with the amino acid sequence composition shown in the GenBank accession number AFT 91381.1.