CN117143748A - Strain with high yield of FR901379 and construction method and application thereof - Google Patents

Strain with high yield of FR901379 and construction method and application thereof Download PDF

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CN117143748A
CN117143748A CN202210570328.1A CN202210570328A CN117143748A CN 117143748 A CN117143748 A CN 117143748A CN 202210570328 A CN202210570328 A CN 202210570328A CN 117143748 A CN117143748 A CN 117143748A
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coleophoma
strain
monooxygenase
cytochrome
leu
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吕雪峰
门萍
黄雪年
谢丽
周宇
王敏
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Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
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Abstract

The application discloses a genetic engineering strain for high-yield micafungin precursor FR901379, which is a genetic engineering strain for over-expressing cytochrome P450 monooxygenase and/or sulfonyl transferase, and an original strain of the genetic engineering strain is a phoma fungus.

Description

Strain with high yield of FR901379 and construction method and application thereof
Technical Field
The application belongs to the technical field of genetic engineering, and in particular relates to a genetic engineering bacterium of the genus Phoma which overexpresses a target protein to improve the yield of FR901379, and a construction method and application thereof.
Background
Echinocandin antifungal drugs are derivatives of natural products of cyclic lipopeptides, and can selectively inhibit the activity of beta-1, 3 glucan synthase in fungal cell walls so that the fungal cell walls cannot be synthesized, and the fungal cells are cracked and dead. With the aggravation of fungal resistance, the proportion of echinocandins in the global antifungal drug market is in an increasing trend year by year. The echinocandin drugs have unique action mechanism, high safety, small toxic and side effects on human bodies, wide antibacterial spectrum and effectiveness on drug-resistant bacteria. Currently, clinically applied echinocandin antifungal drugs include caspofungin, micafungin and anidulafungin. Among them, since micafungin precursor FR901379 has a sulfonyl group, it has excellent water solubility, thereby improving its bioavailability.
However, in the fermentation process of micafungin precursor FR901379, there are problems of low product concentration, more byproducts and difficult separation, resulting in high micafungin production cost. In addition to the target product FR901379, there is also a byproduct 8 in the fermentation product of Coleophoma sp.mefc009, compound 8 being probably caused by insufficient action of the P450 enzymes McfP and McfS. Metabolic engineering transformation based on synthetic pathway is an effective strategy for improving the yield of target products, and through over-expressing key speed-limiting enzyme in the synthetic pathway, the conversion of intermediates to the final products can be promoted, so that the quality improvement and efficiency improvement of the target final products are realized.
Disclosure of Invention
The invention provides a genetically engineered strain, and an original strain of the genetically engineered strain is a phoma fungus.
The phoma fungi include Coleophoma sp.
In a specific embodiment, the fungus of the genus Sphingomyces is Sphingomyces (Coleophoma sp.) meFC009, which is deposited at China general microbiological culture Collection center (CGMCC), with a deposit number of CGMCC No.21058, a deposit date of 2020, 11 months and 18 days, address: the institute of microbiology, national institute of sciences, no. 3, national center for sciences, north chen, west way 1, region of korea, beijing city: 010-64807355.
In other embodiments, the Coleophoma empetri is Coleophoma empetri F-11899.
In the invention, the cytochrome P450 monooxygenase McfP and the sulfonyl transferase McfS in the phoma sphaerocarpum (Coleophoma sp.) MEFC009 are genetically modified to obtain different genetically engineered strains, and the genetically engineered strains can be used for high-yield micafungin precursor FR901379.
In the invention, mcfP is cytochrome P450 monooxygenase (cytochrome P450 monooxygenase), the amino acid sequence of which is shown as SEQ ID No.2, and the nucleic acid sequence of which is shown as SEQ ID No. 1. The McfS is a sulfonyl transferase, the amino acid sequence of the McfS is shown as SEQ ID No.4, and the nucleic acid sequence of the McfS is shown as SEQ ID No. 3.
In the present invention, the cytochrome P450 monooxygenase is also called a P450 enzyme.
In one aspect, the invention provides a cytochrome P450 monooxygenase. In one embodiment, the cytochrome P450 monooxygenase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity compared to SEQ ID No. 2; preferably, the cytochrome P450 monooxygenase is derived from a fungus of the genus phophoma, e.g., coleophoma sp. More preferably, the amino acid sequence of the cytochrome P450 monooxygenase has at least 70% sequence identity compared to SEQ ID No.2, and the cytochrome P450 monooxygenase is derived from a fungus of the genus Phosphaeria. The phoma fungi include Coleophoma sp.or Coleophoma empetri, for example, coleophoma sp.sphaeroides MEFC009. In other embodiments, the Coleophoma empetri is Coleophoma empetri F-11899.
In a preferred embodiment, the amino acid sequence of the cytochrome P450 monooxygenase is shown in SEQ ID No. 2.
In one aspect, the invention provides a sulfonyltransferase. In one embodiment, the sulfonyltransferase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity as compared to SEQ ID No. 4; preferably, the sulfotransferase is derived from a fungus of the genus phoma, e.g., coleophoma sp.or Coleophoma empetri; more preferably, the amino acid sequence of the sulfonyltransferase has at least 70% sequence identity compared to SEQ ID No.4, and the sulfonyltransferase is derived from a fungus of the genus Phosphaerella. The phoma fungi include Coleophoma sp.or Coleophoma empetri, for example, coleophoma sp.sphaeroides MEFC009. In other embodiments, the Coleophoma empetri is Coleophoma empetri F-11899.
In a preferred embodiment, the amino acid sequence of the sulfonyltransferase is as shown in SEQ ID No. 4.
In another aspect, the present invention also provides a biological material comprising the cytochrome P450 monooxygenase or sulfonyltransferase described above or a gene encoding the same. The biological material is selected from the group consisting of: a vector comprising the above cytochrome P450 monooxygenase or sulfonyltransferase, or a host cell comprising the above cytochrome P450 monooxygenase or sulfonyltransferase.
In another aspect, the invention also provides a gene encoding the cytochrome P450 monooxygenase or sulfonyltransferase described above.
In another aspect, the invention also provides a vector comprising the above gene, or a host cell comprising the vector.
In one embodiment, the vectors include cloning vectors and expression vectors, for example, pET-series vectors (e.g., pET-14, pET-21, pET-22, pET-28, pET-30, pET-42, pET-GST, pET-His, pET-Trx, pET-GST, pET-CKS, pET-DsbA), pMAL-series vectors (e.g., pMAL-2C), pGEX-series vectors (e.g., pGEX-4T-2, pGEX-6T-1), pBAD-series vectors (e.g., pBAD-His, pBAD-Myc), pMBP-series vectors (pMBP-P, pMBP-C), pTYB2, pQE-9, pACYCDuet-1, pCDFDuet-1, pColADuet-1, pRSFDuet-1, plP-OmpA, pUC-series vectors (e.g., pUC18, pUC 19), pQE-30, pXH-1, pT pXH-9543, RII 7.
In one embodiment, the host cell is selected from the group consisting of E.coli (e.g., E.coli DH 5. Alpha., E.coli BL21 (DE 3), rosetta (DE 3), codon Plus (DE 3) -RIPL, BL21 Codon Plus (DE 3), top10, JM 109), yeast (e.g., saccharomyces cerevisiae, pichia pastoris, yarrowia lipolytica), phoma sheath, and Neumkang producing bacteria (Glarea lozoyensis).
In another aspect, the invention also provides the use of the cytochrome P450 monooxygenase, the coding gene thereof, the vector containing the gene, the host cell or the biological material in the preparation of the micafungin precursor FR 901379.
In another aspect, the invention also provides the use of the above-described sulfotransferase, its encoding gene, a vector comprising the gene, the above-described host cell, or the above-described biological material in the preparation of micafungin precursor FR 901379.
On the other hand, the invention also provides application of the cytochrome P450 monooxygenase, the coding gene thereof, a vector containing the gene, the host cell or the biological material in preparing a genetic engineering strain of a high-yield micafungin precursor FR 901379; preferably, the original strain of the genetically engineered strain is a phoma fungus.
In another aspect, the invention also provides the application of the sulfonyl transferase, the coding gene thereof, the vector containing the gene, the host cell or the biological material in preparing the genetic engineering strain of the high-yield micafungin precursor FR 901379; preferably, the original strain of the genetically engineered strain is a phoma fungus.
The phoma fungi include Coleophoma sp.
In a specific embodiment, the fungus of the genus Sphingomyces is Sphingomyces (Coleophoma sp.) meFC009, which is deposited at China general microbiological culture Collection center (CGMCC), with a deposit number of CGMCC No.21058, a deposit date of 2020, 11 months and 18 days, address: the institute of microbiology, national institute of sciences, no. 3, national center for sciences, north chen, west way 1, region of korea, beijing city: 010-64807355.
In other embodiments, the Coleophoma empetri is Coleophoma empetri F-11899.
The genetic engineering strain for preparing the high-yield micafungin precursor FR901379 is prepared by introducing the cytochrome P450 monooxygenase and/or sulfonyl transferase into a starting strain; preferably, the introduction is over-expression.
The "introduction" includes the step of expressing, preferably overexpressing, the above-mentioned gene of interest in the starting strain. For example, the gene of interest is constructed on an expression vector, which is transferred into a host cell to express the gene of interest, preferably over-expressed. In other embodiments, the "introducing" comprises inserting the gene of interest into the genome of the host cell; preferably, the insertion into the genome of the host cell may be by homologous recombination double crossover; in one embodiment, insertion of the gene of interest into the appropriate genomic location may be accomplished by inserting the gene of interest and the homology arms into the vector, and then transferring the vector into the host cell, using the homology arms to double-exchange homologous recombination with the host cell genome; in other embodiments, gene editing may also be employed, for example, using a CRISPR/Cas system to cleave at a desired genomic site, while inserting the gene of interest as an exogenous donor into the cleavage site.
On the other hand, the invention also provides application of the genetic engineering strain in the production of micafungin precursor FR 901379.
In another aspect, the present invention also provides a method for preparing micafungin precursor FR901379, comprising the step of fermenting using the genetically engineered strain described above; optionally, the method further comprises the step of isolating/purifying FR 901379.
In the invention, FR901379 is a micafungin precursor, and the structural formula of the micafungin precursor is shown as formula (I):
on the other hand, the invention also provides a genetic engineering bacterium for high-yield micafungin precursor FR901379, wherein the genetic engineering bacterium is a genetic engineering bacterium for over-expressing the cytochrome P450 monooxygenase and/or the sulfonyl transferase, and an original strain of the genetic engineering bacterium is a phoma sphaeroides fungus.
In one embodiment, the cytochrome P450 monooxygenase is overexpressed in the genetically engineered bacterium; the cytochrome P450 monooxygenase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 2; preferably, the cytochrome P450 monooxygenase is derived from a fungus of the genus phophoma; more preferably, the amino acid sequence of the cytochrome P450 monooxygenase has at least 70% sequence identity compared to SEQ ID No.2, and the cytochrome P450 monooxygenase is derived from a fungus of the genus Phosphaeria.
In one embodiment, the sulfonyltransferase is overexpressed in the genetically engineered bacterium; the sulfotransferase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity compared to SEQ ID No. 4; preferably, the sulfotransferase is derived from a fungus of the genus Phoma; more preferably, the amino acid sequence of the sulfonyltransferase has at least 70% sequence identity compared to SEQ ID No.4, and the sulfonyltransferase is derived from a fungus of the genus Phosphaerella.
In one embodiment, the cytochrome P450 monooxygenase and the sulfonyltransferase are simultaneously overexpressed in the genetically engineered bacterium.
The expression of the target gene is higher than that of the wild-type starting strain. In one embodiment, the above overexpression may be achieved by introducing an expression vector to overexpress the gene of interest; in other embodiments, the above overexpression can also be achieved by introducing additional copies of the gene of interest into the starting strain, by increasing the copy number of the gene of interest; in other embodiments, the target gene may be overexpressed by optimizing the promoter of the target gene, for example, by replacing the original promoter of the target gene with a promoter having higher promoter activity.
In another aspect, the present invention also provides a method for preparing/producing micafungin precursor FR901379 by using the above genetically engineered strain, the method comprising the step of culturing the above genetically engineered strain; or the application of the genetically engineered strain in preparing/producing micafungin precursor FR 901379.
In another aspect, the present invention also provides a method for preparing/producing micafungin precursor FR901379, comprising the step of culturing using the above genetically engineered strain; preferably, the method further comprises the step of isolating/purifying FR 901379.
On the other hand, the invention also provides a construction method of the genetically engineered bacterium.
Drawings
FIG. 1 is a result of genome PCR verification of a transformant obtained by knocking out mcfP gene; wherein # 6, # 8 and # 9 are transformants deleted of the gene mcfP, and WT-1 is the control strain Coleophoma sp. - Δku80.
FIG. 2 is the HPLC analysis result of the gene mcfP deletion strain Coleophoma sp. - Δmcfp fermentation product; wherein Coleophoma sp. - Δmcfp is a gene mcfP deleted strain, and WT-1 is Coleophoma sp. - Δku80.
FIG. 3 shows the results of LC-MS analysis of compounds 4, 5, 6, 7 and 8, wherein A is compound 4, B is compound 5, C is compound 6,D, and E is compound 8.
FIG. 4 is a structure of compounds 4, 5, 6, 7 and 8.
FIG. 5 is a result of genome PCR verification of transformants obtained by knocking out mcfS gene; wherein # 1, # 3 and # 7 are transformants deleted of the gene mcfS, and WT-1 is the control strain Coleophoma sp. - Δku80.
FIG. 6 is the results of HPLC analysis of the gene mcfS deleted strain Coleophoma sp. - Δmcfs fermentation product; wherein Coleophoma sp. - Δmcfs is a gene mcfS deleted strain, and WT-1 is Coleophoma sp. - Δku80.
FIG. 7 shows the results of LC-MS analysis of Compound 9.
FIG. 8 is a genomic PCR validation of transformants obtained by over-expression of the P450 enzyme McfP; wherein 1-9 are transformants and WT is wild-type Coleophoma sp.MEFC009.
FIG. 9 shows the HPLC analysis results of engineering strains and Coleophoma sp.MEFC009 fermentation products over-expressing the P450 enzyme McfP; 1: FR901379; WT: coleophoma sp.mefc009; coleophoma sp.: a mutant that overexpresses mcfP.
FIG. 10 is an analysis of FR901379 production in engineering strains and Coleophoma sp.MEFC009 fermentation broth over-expressing the P450 enzyme McfP; WT: coleophoma sp.mefc009; coleophoma sp.: a mutant that overexpresses mcfP. FIG. 11 is a genomic PCR validation of a transformant obtained by overexpressing the sulfonyltransferase McfS; wherein 1-6 are transformants and WT is wild-type Coleophoma sp.MEFC009.
FIG. 12 is the HPLC analysis result of engineering strain and Coleophoma sp.MEFC009 fermentation product over-expressing the sulfonyltransferase McfS; 1: FR901379; WT: coleophoma sp.mefc009; coleophoma sp.: a mutant that overexpresses mcfPS.
FIG. 13 is an analysis of FR901379 production in an engineering strain overexpressing the sulfonyltransferase McfS and Coleophoma sp.MEFC009 fermentation broth; WT: coleophoma sp.mefc009; coleophoma sp.: a mutant that overexpresses mcfPS.
FIG. 14 is a genomic PCR validation of a transformant obtained by simultaneously overexpressing the P450 enzyme McfP and the sulfonyltransferase McfS; wherein 1-8 are transformants and WT is wild-type Coleophoma sp.MEFC009; a: verifying mcfP; b: verifying mcfS;
FIG. 15 is an analysis of FR901379 production in engineering strains and Coleophoma sp.MEFC009 fermentation broth over-expressing the P450 enzymes McfP and the sulfotransferase McfS simultaneously; WT: coleophoma sp.mefc009; coleophoma sp.: mcfP:: mcfS: mutant strains that overexpress mcfP and mcfS.
Detailed Description
The invention will be further illustrated with reference to specific examples, but the invention is not limited to the examples. The materials, reagents, instruments and methods used in the examples below, without any particular description, are conventional in the art and are commercially available.
In this embodiment, the starting strain used is a fungus of the genus Sphaerotheca, the genus Sphaerotheca (Coleophoma sp.) meFC009, and the strain is deposited in China general microbiological culture Collection center (CGMCC), with a deposition number of CGMCC No.21058, a deposition date of 2020, 11 months and 18 days, and an address: the institute of microbiology, national institute of sciences, no. 3, national center for sciences, north chen, west way 1, region of korea, beijing city: 010-64807355.
In the invention, plasmid Mini Kit I reagent (D6942-01) of OMEGA company is adopted for plasmid extraction, and a Cycle-Pure Kit (D6492-01) is adopted for PCR fragment purification by DNA fragment recovery of OMEGA company. One-step cloning of enzymesUltra One Step Cloning Kit from Vazyme, nanjing.
Seed culture medium: 15g/L soluble starch, 10g/L sucrose, 5g/L cottonseed cake powder, 10g/L peptone and 1g/L KH 2 PO 4 ，2g/L CaCO 3 ，pH 6.0-8.0。
Fermentation medium: 30g/L corn starch, 30g/L peptone, 6g/L (NH) 4 ) 2 SO 4 ，1g/L KH 2 PO 4 ，0.3g/L FeSO 4 ·7H 2 O，0.01g/L ZnSO 4 ·7H 2 O，2g/L CaCO 3 ，pH 6.0-8.0。
STC:1M sorbitol, 50mM Tris-HCl (pH 8.0), 50mM CaCl 2 。
PSTC:40% PEG4000,1M sorbitol, 50mM Tris-HCl(pH 8.0)，50mM CaCl 2 。
Top agar: PDB, 1M sorbitol and 4g/L agarose, and incubating at 45-48 ℃ after sterilization.
Regeneration screening media PDA-SH: PDA plate, 1M sorbitol and 100mg/L hygromycin B.
Screening media PDA-H: PDA plates and 100mg/L hygromycin B.
Plasmid pXH-1 is described in Xuenian Huang, xuefang Lu, jian-Jun Li.cloning, characterization and application of a glyceraldehyde-3-phosphate dehydrogenase promoter from Aspergillus terreus, J Ind Microbiol Biotechnol (2014) 41:585-592.
Plasmid pPM-3 is described in Ping Men, min Wang, jinda Li, xuenian Huang, xuefang Lu. Estabishing an efficient genetic manipulation system for sulfated echinocandin producing fungus Coleophoma emertri. Front in microbiology 2021,12,734780.
Example 1 construction of an engineering strain Coleophoma sp.—Δmcfp with the mcfP Gene knocked out
PCR amplification was performed using the genome of wild-type Coleophoma sp.MEFC009 as a template, pfu DNA polymerase (Fermentas, catalog No.: EP 0501), and the upstream sequence U-mcfP of mcfP having a size of about 1.2kb was amplified using primers Umcfp-F (5'-tctcaaggagataactcccacac-3') and Umcfp-R (5'-ctttacgcttgcgatcccgaaTCATTGGGATTGATGCGGATGATAGG-3'), and the downstream sequence D-mcfP having a size of 1.2kb was amplified using primers Dmcfp-F (5'-ccctgggttcgcaaagataattgCGTATCTTTCCACTAATACTGC-3') and Dmcfp-R (5'-caccgtacctgaatcctcat-3'). PCR amplification was performed using the plasmid pXH-1 as a template and primers hph-F (5'-ttcgggatcgcaagcgtaaag-3') and hph-R (5'-caattatctttgcgaacccagg-3') to obtain a hygromycin resistance selection fragment hph of about 2.2kb in size; the hph fragment, the upstream sequence U-mcfP and the downstream sequence D-mcfP are fused by fusion PCR, and then nest primers Umcfp-CS-F (5'-ggacaacgaatagctaaatgaaga-3') and Dmcfp-CS-R (5'-gctctgctattcataactcg-3') are used for amplifying a knockout targeting element Umcfp-hph-Dmcfp with a size of 4.4kb by PCR by taking the fusion product as a template. The mcfP gene sequence is shown as SEQ ID No.1, and the amino acid sequence of McfP is shown as SEQ ID No. 2.
Taking Coleophoma sp. -delta ku80 as a starting strain, firstly taking a small amount of hypha from a PDA flat plate, crushing by using a handheld homogenizer, taking 1mL of seed liquid, inoculating into 50mL of seed culture medium, and carrying out shake culture at 220rpm and 25 ℃ in a 250mL triangular flask. After 2 days, mycelia were collected by centrifugation. 5000rpm,4℃for 5min. The mycelium is crushed again by a homogenizer, 0.5mL-2mL of seed liquid is inoculated to 50mL of seed culture medium, the culture is carried out for 1 day under the same condition, the culture medium and the mycelium are poured into a 50mL sterile centrifuge tube together, the speed is 5000rpm, and the mycelium is collected by centrifugation. With 0.6M MgSO 4 The mycelium was washed 2 times. 1g of mycelium is weighed, 10mL of enzymolysis liquid is added, and the mixture is treated for 1 to 4 hours at 30 ℃ and 100 rpm. The enzymolysis liquid comprises the following components: 1% cellulase, 0.6% lywallzyme, 0.6% snailase, 0.6M MgSO 4 The bacteria were filtered through a sterile filter of 0.22 μm. The protoplast reaction solution was filtered through a sterile magic filter cloth. Protoplasts were collected by centrifugation at 5000rpm at 4 ℃. Washing with ice-chilled STC once, re-suspending the protoplasts in the chilled STC, and adjusting the protoplast concentration to 5X 10 with STC 7 And (3) obtaining protoplast suspension at a ratio of one mL to the other mL.
To 140. Mu.L of the protoplast suspension, 10. Mu.L of Umcfp-hph-Dmcfp fragment was added, followed by 50. Mu.L of PSTC, gently mixed, and ice-bathed for 30min. Adding 1mL of PSTC, uniformly mixing, and standing at room temperature for 20min; then mixed with 10mL of top agar, poured onto 3 regeneration screening culture medium plates PDA-SH, and cultured for 5-7 days under dark conditions at 30 ℃ to obtain transformants.
Transformants with hygromycin resistance were selected from the transformation screening plates and transferred to PDA-H, and subcultured at 25℃for 4-6 days for serial passage for 3 passages. Selecting 3 transformants (No. 6, no. 8 and No. 9) with stable passage for monospore separation and purification, and extracting the genome of the transformant after monospore separation. PCR verification of the transformant genome using the external primers Umcfp-F (5'-tctcaaggagataactcccacac-3') and Dmcfp-R (5'-caccgtacctgaatcctcat-3') allowed the amplification of positive transformants with a band size of about 4.6kb, whereas Coleophoma sp.—Δku80 could only amplify a band size of about 2.9kb, FIG. 1 illustrates that the 6#,8#,9# transformants were positive transformants, indicating that homologous recombination occurred at the position of the gene mcfP, integrating the exogenous fragment Umcfp-hph-Dmcfp.
EXAMPLE 2 fermentation and product analysis of mcfP Gene-deleted engineering Strain Coleophoma sp. -Deltamcfp
3 mcfP gene-deleted engineering strains Coleophoma sp.—Δmcfp6#, 8#,9# and a control strain Coleophoma sp.—Δku80 were inoculated on PDA solid plates and cultured at 25 ℃ for 4-6 days. Selecting a small amount of mycelium, and extracting with a nucleic acid extractor-24) breaking the mycelium, inoculating the broken mycelium into 50mL of seed culture medium (250 mL triangular flask) of Coleophoma sp. At 25 ℃,220rpm, and shaking culture for 48h. The seed solution was shake-cultured at 25℃and 220rpm for 8 days with 5mL of a fermentation medium of Coleophoma sp, and 3 strains were placed in parallel. 1mL of each bottle of fermentation broth is taken, an equal volume of methanol is added, ultrasonic extraction is carried out for 1h, and the supernatant is taken after centrifugation. The treated samples were filtered with a 0.22 μm organic filter and analyzed by HPLC and LC-MS.
The HPLC analysis method comprises the following steps: the liquid chromatographic column is Agilent C-18 reverse column 883975-902 (4.6X150 mm,5 μm); the mobile phase is A:0.05% (volume ratio) aqueous trifluoroacetic acid, mobile phase B:0.05% (volume ratio) acetonitrile trifluoroacetic acid solution, flow rate of 1mL/min, ultraviolet detection wavelength: 210nm,30℃and a total elution time of 37min. Gradient elution conditions: and the mobile phase B is linearly increased from 5% to 24% by volume of the mobile phase for 0-5min, the mobile phase B is linearly increased from 24% to 62% by volume of the mobile phase for 5-35min, and the mobile phase B is linearly increased from 62% to 100% by volume of the mobile phase for 35-37 min. The results are shown in FIG. 2; in comparison with the starting strain Coleophoma sp.—Δku80, compounds 1, 2, 3 disappeared, and a corresponding further 5 compounds with the same uv absorption as compounds 1, 2, 3 appeared. Further, the compounds 4, 5, 6, 7 and 8 were isolated and purified, and analyzed by liquid chromatography-mass spectrometry (LC-MS) and Nuclear Magnetic Resonance (NMR). The LC-MS analysis method comprises the following steps: agilent 1290 high performance liquid chromatography, chromatographic columnA C18 column (2.1X105 mm,1.8 μm) of Agilent Zorbax Extend-C18; the total flow rate of the mobile phase is 0.6mL/min; mobile phase a:0.05% (volume ratio) aqueous formic acid, mobile phase B:0.05% (volume ratio) acetonitrile formate solution, total elution time 7.0min; the elution conditions were: gradient elution conditions: and the mobile phase B is linearly increased from 5% to 20% by volume of the mobile phase for 0-1min, the mobile phase B is linearly increased from 20% to 60% by volume of the mobile phase for 1-6min, and the mobile phase B is linearly increased from 60% to 100% by volume of the mobile phase for 6-7 min. The results are shown in FIG. 3; as a result of the NMR analysis, it was found that 4 (formula: C 51 H 82 N 8 O 17 Theoretical value: [ M+H ]]+1079.5871, actual value: 1079.5873 5 (molecular formula: c (C) 50 H 80 N 8 O 16 Theoretical value: [ M+H ]] + 1049.5765, actual value: 1049.5766 6 (molecular formula: c (C) 51 H 82 N 8 O 16 Theoretical value: [ M+H ]] + 1063.5922, actual 1063.5921), 7 (molecular formula: c (C) 51 H 82 N 8 O 15 Theoretical value: [ M+H ]] + 1047.5972, actual value: 1047.5969 And 8 (formula: c (C) 51 H 82 N 8 O 14 Theoretical value: [ M+H ]] + 1031.6023, actual value: 1031.6022 These intermediates were presumed to be less in oxysulfonyl and some hydroxyl groups based on molecular weight. The structures of compounds 4, 5, 6, 7 and 8 were further identified by NMR. As shown in fig. 4, they share a common feature: the disappearance of the oxysulfonyl group indicates that cytochrome P450 monooxygenase encoded by mcfP is responsible for the first step of hydroxylation of the C3' position of the L-homotyrosine benzene ring formed by the oxysulfonylation module in the FR901379 structure.
Example 3 construction of an engineering strain Coleophoma sp.—Δmcfs with knockout of mcfS Gene
PCR amplification was performed using the genome of wild-type Coleophoma sp. With pfu DNA polymerase (Fermentas, catalog No.: EP 0501), with primers Umcfs-F (5'-gcgccttcgaagcgggcaac-3') and Umcfs-R (5'-ctttacgcttgcgatcccgaaTCGAAGGCCTCTTTCCACAAC-3') to obtain an upstream sequence U-mcfS of approximately 1.2kb mcfS, and with primers Dmcfs-F (5'-cctgggttcgcaaagataattgACATATTCAAGTACAGCCCCC-3') and Dmcfs-R (5'-tagtccagaggatgacttcc-3') to obtain a downstream sequence D-mcfS of 1.2kb mcfS. PCR amplification was performed using the plasmid pXH-1 as a template and primers hph-F (5'-ttcgggatcgcaagcgtaaag-3') and hph-R (5'-caattatctttgcgaacccagg-3') to obtain a hygromycin resistance selection fragment hph of about 2.2kb in size; the hph fragment, the upstream sequence U-mcfS and the downstream sequence D-mcfS are fused by fusion PCR, and then nest primers Umcfs-CS-F (5'-gaatactttgctcgcaggtg-3') and Dmcfs-CS-R (5'-gccaatctataaagggaaagg-3') are used for amplifying a knockout targeting element Umcfs-hph-Dmcfs with a size of 4.4kb by PCR by taking the fusion product as a template.
Taking Coleophoma sp. -delta ku80 as a starting strain, firstly taking a small amount of hypha from a PDA flat plate, crushing by using a handheld homogenizer, taking 1mL of seed liquid, inoculating into 50mL of seed culture medium, and carrying out shake culture at 220rpm and 25 ℃ in a 250mL triangular flask. After 2 days, mycelia were collected by centrifugation. 5000rpm,4℃for 5min. The mycelium is crushed again by a homogenizer, 0.5mL-2mL of seed liquid is inoculated to 50mL of seed culture medium, the culture is carried out for 1 day under the same condition, the culture medium and the mycelium are poured into a 50mL sterile centrifuge tube together, the speed is 5000rpm, and the mycelium is collected by centrifugation. With 0.6M MgSO 4 The mycelium was washed 2 times. 1g of mycelium is weighed, 10mL of enzymolysis liquid is added, and the mixture is treated for 1 to 4 hours at 30 ℃ and 100 rpm. The enzymolysis liquid comprises the following components: 1% cellulase, 0.6% lywallzyme, 0.6% snailase, 0.6M MgSO 4 The bacteria were filtered through a sterile filter of 0.22 μm. The protoplast reaction solution was filtered through a sterile magic filter cloth. Protoplasts were collected by centrifugation at 5000rpm at 4 ℃. Washing with ice-chilled STC once, re-suspending the protoplasts in the chilled STC, and adjusting the protoplast concentration to 5X 10 with STC 7 And (3) obtaining protoplast suspension at a ratio of one mL to the other mL.
To 140. Mu.L of the protoplast suspension, 10. Mu.L of Umcfs-hph-Dmcfs fragment was added, followed by 50. Mu.L of PSTC, gently mixed, and ice-bathed for 30min. Adding 1mL of PSTC, uniformly mixing, and standing at room temperature for 20min; then mixed with 10mL of top agar, poured onto 3 regeneration screening culture medium plates PDA-SH, and cultured for 5-7 days under dark condition at 30 ℃ to obtain transformants.
Transformants with hygromycin resistance were selected from the transformation screening plates and transferred to PDA-H, and subcultured at 25℃for 4-6 days for serial passage for 3 passages. Selecting 3 transformants (No. 1, no. 3 and No. 7) with stable passage for monospore separation and purification, and extracting the genome of the transformant after monospore separation. PCR verification of the transformant genome using the external primers Umcfs-F (5'-gcgccttcgaagcgggcaac-3') and Dmcfs-R (5'-tagtccagaggatgacttcc-3') allowed the amplification of positive transformants with a band size of about 4.9kb, whereas Coleophoma sp.—Deltaku 80 could only amplify a band size of about 3.1kb, FIG. 5 illustrates that the 1#,3#,7# transformants were positive transformants, indicating that homologous recombination occurred at the location of the gene mcfS, integrating the exogenous fragment Umcfs-hph-Dmcfs.
EXAMPLE 4 fermentation and product analysis of mcfS Gene-deleted engineering Strain Coleophoma sp. -Deltamcfs
3 mcfS gene deletion engineering strains Coleophoma sp.—DeltamcfS1#, 3#,7# and a control strain Coleophoma sp.—Deltaku80 were inoculated on a PDA solid plate and cultured at 25 ℃ for 4-6 days. Selecting a small amount of mycelium, and extracting with a nucleic acid extractor-24) breaking the mycelium, inoculating the broken mycelium into 50mL of seed culture medium (250 mL triangular flask) of Coleophoma sp. At 25 ℃,220rpm, and shaking culture for 48h. The seed solution was shake-cultured at 25℃and 220rpm for 8 days with 5mL of a fermentation medium of Coleophoma sp, and 3 strains were placed in parallel. 1mL of each bottle of fermentation broth is taken, an equal volume of methanol is added, ultrasonic extraction is carried out for 1h, and the supernatant is taken after centrifugation. The treated samples were filtered with a 0.22 μm organic filter and analyzed by HPLC and LC-MS.
The HPLC analysis method comprises the following steps: the liquid chromatographic column is Agilent C-18 reverse column 883975-902 (4.6X150 mm,5 μm); the mobile phase is A:0.05% (volume ratio) aqueous trifluoroacetic acid, mobile phase B:0.05% (volume ratio) acetonitrile trifluoroacetic acid solution, flow rate of 1mL/min, ultraviolet detection wavelength: 210nm,30℃and a total elution time of 37min. Gradient elution conditions: 0-5min, flowingThe volume of the phase B in the mobile phase is linearly increased from 5% to 24%,5-35min, the volume of the mobile phase B in the mobile phase is linearly increased from 24% to 62%,35-37min, and the volume of the mobile phase B in the mobile phase is linearly increased from 62% to 100%. The results are shown in FIG. 6; compounds 1, 2, 3 disappeared compared to the starting strain Coleophoma sp.—Δku80, yielding small amounts of compounds 6, 7, 8 and 9. The Coleophoma sp.—Δmcfs fermentation product was analyzed by LC-MS. The LC-MS analysis method comprises the following steps: high Performance Liquid Chromatography (HPLC) of Agilent 1290, column Agilent Zorbax Extend-C18 (2.1X105 mm,1.8 μm); the total flow rate of the mobile phase is 0.6mL/min; mobile phase a:0.05% (volume ratio) aqueous formic acid, mobile phase B:0.05% (volume ratio) acetonitrile formate solution, total elution time 7.0min; gradient elution conditions: and the mobile phase B is linearly increased from 5% to 20% by volume of the mobile phase for 0-1min, the mobile phase B is linearly increased from 20% to 60% by volume of the mobile phase for 1-6min, and the mobile phase B is linearly increased from 60% to 100% by volume of the mobile phase for 6-7 min. The results are shown in FIG. 7; as a result of LC-MS analysis, the sulfonyl group in the FR901379 structure disappeared when the gene mcfS was knocked out, resulting in compounds 6, 7, 8 and 9. Compounds 6, 7 and 8 are also present in the knockout strain Coleophoma sp. - Δmcfp, these 3 compounds share a common feature, with the oxysulfonyl group at the C3' position of the L-homotyrosine benzene ring being deleted. Compound 9 was analyzed by LC-MS, compound 9 formula: c (C) 51 H 82 N 8 O 18 Theoretical value: [ M+H ]] + 1095.5820 actual value 1095.5823, molecular weight 80 lower than that of Compound 1, presumably shows fewer sulfonyl groups (SO 3 - ) The method comprises the steps of carrying out a first treatment on the surface of the Further, it was confirmed by NMR that Compound 9 had only a hydroxyl group at the C3' position of the L-homotyrosine benzene ring. The above results indicate that mcfS is responsible for transferring the sulfonyl group to the hydroxyl group at the C3' position of the L-homotyrosine benzene ring in FR901379 biosynthesis. The mcfS gene sequence is shown in SEQ ID No.3, and the McfS amino acid sequence is shown in SEQ ID No. 4.
EXAMPLE 5 construction of recombinant strains overexpressing the P450 enzyme McfP
5.1 construction of the expression cassette for the overexpression of the P450 enzyme McfP
PCR amplification was performed using the plasmid pXH-1 as a template and the primers PgpdAT-F (5'-ccctgggttcgcaaagataattggttacactctgggaggatcc-3') and PgpdAT-R (5'-gttgtgatgattgatgagttg-3') to obtain a promoter fragment PgpdAT of about 0.7kb in size; PCR amplification is carried out by using a genome of Coleophoma sp. As a template and using a primer mcfP-F (5 '-aactcatcaatcatcacaacATGATAAATCTTGCAAGTCCCCTC') and mcfP-R (5'-cacaaaattcttcatttatttactaccgatgaccttcaaggac-3') to obtain a fragment mcfP having a size of about 1.8 kb; using plasmid pPM-3 as a template, and using primers Tpgk-F (5'-taaataaatgaagaattttg-3') and Tpgk-R (5'-tacctctaaacaagtgtacc-3') as primers to carry out PCR amplification to obtain a terminator fragment Tpgk with the size of about 0.5 kb; fusion PCR is carried out on the fragments PgpdAT, mcfP and Tpgk, so that the expression cassette PgpdAT-mcfP-Tpgk is obtained. PCR amplification was performed using the plasmid pXH-1 as a template and the primers hph-F (5'-ttcgggatcgcaagcgtaaag-3') and hph-R (5'-caattatctttgcgaacccagg-3') to obtain a hygromycin resistance selection fragment hph of about 2.2kb in size.
5.2 Co-transformation construction of recombinant strains over-expressed by the P450 enzyme McfP
Taking Coleophoma sp.MEFC009 as a starting strain, firstly taking a small amount of mycelium from a PDA flat plate, crushing the mycelium by using a handheld homogenizer, taking 1mL of seed liquid, inoculating the seed liquid into 50mL of seed culture medium, and carrying out shake culture at 220rpm and 25 ℃ in a 250mL triangular flask. After 2 days, mycelia were collected by centrifugation. 5000rpm,4℃for 5min. The mycelium is crushed again by a homogenizer, 0.5mL-2mL of seed liquid is inoculated to 50mL of seed culture medium, the culture is carried out for 1 day under the same condition, the culture medium and the mycelium are poured into a 50mL sterile centrifuge tube together, the speed is 5000rpm, and the mycelium is collected by centrifugation. With 0.6M MgSO 4 The mycelium was washed 2 times. 1g of mycelium is weighed, 10mL of enzymolysis liquid is added, and the mixture is treated for 1 to 4 hours at 30 ℃ and 100 rpm. The enzymolysis liquid comprises the following components: 1% cellulase, 0.6% lywallzyme, 0.6% snailase, 0.6M MgSO 4 The bacteria were filtered through a sterile filter of 0.22 μm. The protoplast reaction solution was filtered through a sterile magic filter cloth. Protoplasts were collected by centrifugation at 5000rpm at 4 ℃. Washing with ice-chilled STC once, re-suspending the protoplasts in the chilled STC, and adjusting the protoplast concentration to 5X 10 with STC 7 Individual/mL, a protoplast suspension is obtained.
To 140. Mu.L of the protoplast suspension, pgpdAT-mcfP-Tpgk fragment and hph fragment were added in a molar ratio of 5:1, and 50. Mu.L of PSTC was added thereto, followed by gentle mixing and ice-bath for 30min. Adding 1mL of PSTC, uniformly mixing, and standing at room temperature for 20min; then mixed with 10mL of top agar, poured onto 3 regeneration screening culture medium plates PDA-SH, and cultured for 5-7 days under dark conditions at 30 ℃ to obtain transformants.
Transformants with hygromycin resistance were selected from the transformation screening plates and transferred to PDA-H, and subcultured at 25℃for 5-7 days for 3 consecutive passages. Selecting 9 transformants for separation and purification, and carrying out PCR verification on the genome of the purified transformants by using primers PgpdAT-F (5'-ccctgggttcgcaaagataattggttacactctgggaggatcc-3') and mcfP-R (5'-cacaaaattcttcatttatttactaccgatgaccttcaaggac-3'), wherein the positive transformants can be amplified by a band with the size of about 2.5kb, as shown in FIG. 8; transformants # 2, # 3, # 6, # 7, # 8 and # 9 have the expression elements PgpdAT-mcfP-Tpgk integrated on their genomes.
Example 6 fermentation verification of recombinant strains overexpressing the P450 enzyme McfP
The mcfP-overexpressing engineered strain obtained in example 5 and the control strain Coleophoma sp.mefc009 were inoculated onto PDA solid plates and incubated at 25 ℃ for 5-7 days. Selecting a small amount of mycelium, and extracting with a nucleic acid extractor -24) breaking the mycelium, inoculating the broken mycelium to 50mL of seed culture medium of Coleophoma sp (250 mL triangular flask), 25 ℃,220rpm, and shaking culture for 45-48 h. The seed solution of the above culture was shake-cultured at 25℃and 220rpm for 8 days with 5mL of fermentation medium of Coleophoma sp. Each strain was set in 3 replicates. 1mL of each bottle of fermentation broth is taken, an equal volume of methanol is added, ultrasonic extraction is carried out for 1h, and the supernatant is taken after centrifugation. The treated sample was filtered with a 0.22 μm organic filter and analyzed by HPLC.
The HPLC analysis method comprises the following steps: the liquid chromatographic column is Agilent C-18 reverse column 883975-902 (4.6X150 mm,5 μm); the mobile phase is A:0.05% (volume ratio) aqueous trifluoroacetic acid, mobile phase B:0.05% (volume ratio) acetonitrile trifluoroacetic acid solution, flow rate of 1mL/min, ultraviolet detection wavelength: 210nm,30℃and a total elution time of 25min. Gradient elution conditions: and the mobile phase B is linearly increased from 5% to 40% by volume of the mobile phase for 0-5min, the mobile phase B is linearly increased from 40% to 62% by volume of the mobile phase for 5-20min, and the mobile phase B is linearly increased from 62% to 100% by volume of the mobile phase for 20-25 min. The HPLC analysis results are shown in FIG. 9, from which it can be seen that the yield of compound FR901379 in mcfP was increased for the engineered strain Coleophoma sp. As shown in FIG. 10, the yield of FR901379 in the mcfP-overexpressing engineering strain was 477mg/L, which was increased by 26.3% compared to the control strain Coleophoma sp.MEFC009.
EXAMPLE 7 construction of recombinant strains overexpressing the Sulfonyl transferase McfS
PCR amplification was performed using the genome of Coleophoma sp.MEFC009 as a template and the primers mcfS-F (5'-aactcatcaatcatcacaacATGGCTTTAGACCGCCAGAATGC-3') and mcfS-R (5'-cacaaaattcttcatttatttactacttcctagctagccaaacag-3') to obtain a fragment mcfS of about 1.0kb in size; fusion PCR was used to fuse the fragments PgpdAT, tpgk and the fragment mcfS of example 5 to obtain the expression cassette PgpdAT-mcfS-Tpgk.
Taking Coleophoma sp.MEFC009 as a starting strain, firstly taking a small amount of mycelium from a PDA flat plate, crushing the mycelium by using a handheld homogenizer, taking 1mL of seed liquid, inoculating the seed liquid into 50mL of seed culture medium, and carrying out shake culture at 220rpm and 25 ℃ in a 250mL triangular flask. After 2 days, mycelia were collected by centrifugation. 5000rpm,4℃for 5min. The mycelium is crushed again by a homogenizer, 0.5mL-2mL of seed liquid is inoculated to 50mL of seed culture medium, the culture is carried out for 1 day under the same condition, the culture medium and the mycelium are poured into a 50mL sterile centrifuge tube together, the speed is 5000rpm, and the mycelium is collected by centrifugation. With 0.6M MgSO 4 The mycelium was washed 2 times. 1g of mycelium is weighed, 10mL of enzymolysis liquid is added, and the mixture is treated for 1 to 4 hours at 30 ℃ and 100 rpm. The enzymolysis liquid comprises the following components: 1% cellulase, 0.6% lywallzyme, 0.6% snailase, 0.6M MgSO 4 The bacteria were filtered through a sterile filter of 0.22 μm. The protoplast reaction solution is treated withFiltration was performed through sterile magic filter cloth. Protoplasts were collected by centrifugation at 5000rpm at 4 ℃. Washing with ice-chilled STC once, re-suspending the protoplasts in the chilled STC, and adjusting the protoplast concentration to 5X 10 with STC 7 And (3) obtaining protoplast suspension at a ratio of one mL to the other mL.
To 140. Mu.L of the protoplast suspension, pgpdAT-mcfS-Tpgk fragment and hph fragment were added, 50. Mu.L of PSTC was added, and the mixture was gently mixed and ice-cooled for 30min. Adding 1mL of PSTC, uniformly mixing, and standing at room temperature for 20min; then mixed with 10mL of top agar, poured onto 3 regeneration screening culture medium plates PDA-SH, and cultured for 5-7 days under dark conditions at 30 ℃ to obtain transformants.
Transformants with hygromycin resistance were selected from the transformation screening plates and transferred to PDA-H, and subcultured at 25℃for 5-7 days for 3 consecutive passages. Selecting 6 transformants for separation and purification, and carrying out PCR verification on the genome of the purified transformants by using primers PgpdAT-F (5'-ccctgggttcgcaaagataattggttacactctgggaggatcc-3') and mcfS-R (5'-cacaaaattcttcatttatttactacttcctagctagccaaacag-3'), wherein the positive transformants can be amplified by a band with the size of about 1.7kb, as shown in FIG. 11; all of these 6 transformants were positive transformants, and the expression element PgpdAT-mcfS-Tpgk was integrated on the genome.
Example 8 fermentation verification of recombinant strains overexpressing Sulfonyl transferase McfS
The engineering strain overexpressing the sulfonyltransferase McfS of example 7 and the control strain Coleophoma sp.MEFC009 were inoculated onto PDA solid plates and incubated at 25℃for 5-7 days. Selecting a small amount of mycelium, and extracting with a nucleic acid extractor-24) breaking the mycelium, inoculating the broken mycelium into a seed culture medium (250 mL triangular flask) of 50mL Coleophoma sp.MEFC009, and performing shaking culture at 25 ℃ and 220rpm for 45-48 h. The seed solution of the above culture was shake-cultured at 25℃and 220rpm for 8 days with 5mL of fermentation medium of Coleophoma sp. Each strain was set in 3 replicates. Taking 1mL from each bottle of fermentation broth, adding equal volume of methanol, ultrasonically extracting for 1h, centrifuging, and collectingClearing. The treated sample was filtered with a 0.22 μm organic filter and analyzed by HPLC.
The HPLC analysis method comprises the following steps: the liquid chromatographic column is Agilent C-18 reverse column 883975-902 (4.6X150 mm,5 μm); the mobile phase is A:0.05% (volume ratio) aqueous trifluoroacetic acid, mobile phase B:0.05% (volume ratio) acetonitrile trifluoroacetic acid solution, flow rate of 1mL/min, ultraviolet detection wavelength: 210nm,30℃and a total elution time of 25min. Gradient elution conditions: and the mobile phase B is linearly increased from 5% to 40% by volume of the mobile phase for 0-5min, the mobile phase B is linearly increased from 40% to 62% by volume of the mobile phase for 5-20min, and the mobile phase B is linearly increased from 62% to 100% by volume of the mobile phase for 20-25 min. As shown in FIG. 12, the yield of the compound FR901379 in mcfS was increased in the engineering strain Coleophoma sp. As shown in FIG. 13, the yield of FR901379 in the engineering strain over-expressing mcfS was 487.5mg/L, which was 30% higher than that of the control strain Coleophoma sp.MEFC009.
EXAMPLE 9 construction of recombinant strains that overexpress both the P450 enzyme McfP and the Sulfonyl transferase McfS
Taking Coleophoma sp.MEFC009 as a starting strain, firstly taking a small amount of mycelium from a PDA flat plate, crushing the mycelium by using a handheld homogenizer, taking 1mL of seed liquid, inoculating the seed liquid into 50mL of seed culture medium, and carrying out shake culture at 220rpm and 25 ℃ in a 250mL triangular flask. After 2 days, mycelia were collected by centrifugation. 5000rpm,4℃for 5min. The mycelium is crushed again by a homogenizer, 0.5mL-2mL of seed liquid is inoculated to 50mL of seed culture medium, the culture is carried out for 1 day under the same condition, the culture medium and the mycelium are poured into a 50mL sterile centrifuge tube together, the speed is 5000rpm, and the mycelium is collected by centrifugation. With 0.6M MgSO 4 The mycelium was washed 2 times. 1g of mycelium is weighed, 10mL of enzymolysis liquid is added, and the mixture is treated for 1 to 4 hours at 30 ℃ and 100 rpm. The enzymolysis liquid comprises the following components: 1% cellulase, 0.6% lywallzyme, 0.6% snailase, 0.6M MgSO 4 The bacteria were filtered through a sterile filter of 0.22 μm. The protoplast reaction solution was filtered through a sterile magic filter cloth. Protoplasts were collected by centrifugation at 5000rpm at 4 ℃. Washing with ice-chilled STC once, re-suspending the protoplasts in the chilled STCAnd the protoplast concentration was adjusted to 5X 10 with STC 7 And (3) obtaining protoplast suspension at a ratio of one mL to the other mL.
The expression cassettes PgpdAT-mcfP-Tpgk, pgpdAT-mcfS-Tpgk and hph constructed in examples 5 and 7 above were added to 200. Mu.L of the above protoplast suspension, and 50. Mu.L of PSTC was added thereto, gently mixed, and ice-cooled for 30min. Adding 1mL of PSTC, uniformly mixing, and standing at room temperature for 20min; then mixed with 10mL of top agar, poured onto 3 regeneration screening culture medium plates PDA-SH, and cultured for 5-7 days under dark conditions at 30 ℃ to obtain transformants.
Transformants with hygromycin resistance were selected from the transformation screening plates and transferred to PDA-H, and subcultured at 25℃for 5-7 days for 3 consecutive passages. 8 transformants are selected for separation and purification, and PCR verification is carried out on the purified transformant gene groups by using primers PgpdAT-F (5'-ccctgggttcgcaaagataattggttacactctgggaggatcc-3') and mcfP-R (5'-cacaaaattcttcatttatttactaccgatgaccttcaaggac-3') respectively, wherein PgpdAT-F (5'-ccctgggttcgcaaagataattggttacactctgggaggatcc-3') and mcfS-R (5'-cacaaaattcttcatttatttactacttcctagctagccaaacag-3') can simultaneously amplify the positive transformants with the sizes of about 2.5kb and 1.7kb, as shown in FIG. 14; transformants # 2, # 3, # 4, # 5, # 6 and # 8 have both expression elements PgpdAT-mcfP-Tpgk and PgpdAT-mcfS-Tpgk integrated on their genomes.
Example 10 fermentation verification of recombinant strains simultaneously overexpressing the P450 enzyme McfP and the Sulfonyl transferase McfS
The engineering strain and the control strain, coleophoma sp.MEFC009, which simultaneously overexpress the P450 enzymes McfP and McfS in example 9, were inoculated onto PDA solid plates and incubated at 25℃for 5-7 days. Selecting a small amount of mycelium, and extracting with a nucleic acid extractor-24) breaking the mycelium, inoculating the broken mycelium into 50mL of seed culture medium (250 mL triangular flask), 25 ℃,220rpm, and shaking culture for 45-48h. The seed solution obtained in the above-mentioned culture was subjected to shaking culture at 25℃and 220rpm for 8 days in a fermentation medium (250 mL triangular flask) in an amount of 5mL to 50mL, and 3 strains were placed in parallel. From each bottle1mL of fermentation broth is taken, methanol with the same volume is added, ultrasonic extraction is carried out for 1h, centrifugation is carried out, and supernatant is taken. The treated sample was filtered with a 0.22 μm organic filter and analyzed by HPLC. The fermentation result is shown in FIG. 15, and the yield of the engineering strain Coleophoma sp, which simultaneously overexpresses McfP and McfS, is 698mg/L, and is improved by 84% compared with that of the control strain Coleophoma sp.MEFC009.
While the invention has been described in terms of preferred embodiments, it is not intended to limit the invention, but rather, it will be apparent to those skilled in the art that various changes and modifications can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
SEQUENCE LISTING
<110> Qingdao bioenergy and Process institute of China academy of sciences
<120> high-yield FR901379 strain, construction method and application thereof
<130> 11
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1503
<212> DNA
<213> Artificial Sequence
<220>
<223> mcfP
<400> 1
atgataaatc ttgcaagtcc cctcttcgca acaacagcag ttctagtctg gctcagcagt 60
ctcataatct atcgcctata tctctctcca ctatctcgat ttcccggccc aaaactcgct 120
gctctaacag gatggtacga gacatacttc gacctcttta aacggggtcg ctactggatc 180
gagattgaac gcatgcacga agtctatggc cctatcatcc gcatcaatcc caatgagcta 240
catgttaatg acccagaatg gaatgagccc tacaagatca gcggccgcgt tgacaagtat 300
gactggtact acacctttgt tggtagttcc ggatcctcat ctgcattcgg aaccatagac 360
cacgacgttc atcgtggccg ccggaaagct caacagggct atttcaccac cgacgccatc 420
acgcgctttg aaccacattt agaaaccctg acagcaaagt tctgcgcaag actagacggc 480
ttcaagggga cgggaaagca tgttaatctc tccgatgcgt tccgatcaat cgcggtggat 540
gtggccgcga tgtttacatt gaatcaatcg tatggtttca tcgatgaccc ggatttcaag 600
gccgaggtcc atcaagggat ccgggcattt ccggatattg gagtgctgaa tcgccatttt 660
acgggtttgt tcgtggtttt ggagtcaatc catagatggg tgttgagtgt tatcaacccg 720
tcagaagaag ataatgggtt actcacaagt agaataaacc tgcattgtaa agctattatt 780
gccgactacg ccagtaagaa aggcgacgtc aagcccaata tcattcacag aatgctagac 840
gcaccagaac tatcgatgaa agataagaca gcgtggcgcc ttcaattgga ggcgcgcacc 900
cttataggag ctggaactga aacgacagga cacacattag ccgtcatagc attccatctg 960
ctagcaaatc cggagaaggc aaagaggttg aaggaggaga tcttagctac gaaagaaggg 1020
cgggaaaagc ctttaactta tcaggagtta caaatgcttc cgtatttatc ttctgtggtc 1080
cttgaaggtc atcgcatttc tagtgttgta tcaggtcgtc tgccacgggt caatacaaaa 1140
gagccgctca gatatggtga ctatagtatc cctattggca cacccgtcag caccacccaa 1200
cggttaacac actacaatgc caccatattc ccctccccaa acacattcct ccccgaacgt 1260
tggcttcagc cctcggaacg aaagcgcctg gagaaataca tccagccgtt cgggcgtggc 1320
tcaagatctt gtataggcat gcatcttgca aatgcagaga tttacaaaac attggcggag 1380
atgtttgcaa ggtttgacat gaagttatat gatacggagt tcgaggatat tatgcaagtg 1440
catgactttt ttacttcgtt tccatcgagc gagaggggtt taagaatact tgtggaagca 1500
taa 1503
<210> 2
<211> 500
<212> PRT
<213> Artificial Sequence
<220>
<223> mcfP
<400> 2
Met Ile Asn Leu Ala Ser Pro Leu Phe Ala Thr Thr Ala Val Leu Val
1 5 10 15
Trp Leu Ser Ser Leu Ile Ile Tyr Arg Leu Tyr Leu Ser Pro Leu Ser
20 25 30
Arg Phe Pro Gly Pro Lys Leu Ala Ala Leu Thr Gly Trp Tyr Glu Thr
35 40 45
Tyr Phe Asp Leu Phe Lys Arg Gly Arg Tyr Trp Ile Glu Ile Glu Arg
50 55 60
Met His Glu Val Tyr Gly Pro Ile Ile Arg Ile Asn Pro Asn Glu Leu
65 70 75 80
His Val Asn Asp Pro Glu Trp Asn Glu Pro Tyr Lys Ile Ser Gly Arg
85 90 95
Val Asp Lys Tyr Asp Trp Tyr Tyr Thr Phe Val Gly Ser Ser Gly Ser
100 105 110
Ser Ser Ala Phe Gly Thr Ile Asp His Asp Val His Arg Gly Arg Arg
115 120 125
Lys Ala Gln Gln Gly Tyr Phe Thr Thr Asp Ala Ile Thr Arg Phe Glu
130 135 140
Pro His Leu Glu Thr Leu Thr Ala Lys Phe Cys Ala Arg Leu Asp Gly
145 150 155 160
Phe Lys Gly Thr Gly Lys His Val Asn Leu Ser Asp Ala Phe Arg Ser
165 170 175
Ile Ala Val Asp Val Ala Ala Met Phe Thr Leu Asn Gln Ser Tyr Gly
180 185 190
Phe Ile Asp Asp Pro Asp Phe Lys Ala Glu Val His Gln Gly Ile Arg
195 200 205
Ala Phe Pro Asp Ile Gly Val Leu Asn Arg His Phe Thr Gly Leu Phe
210 215 220
Val Val Leu Glu Ser Ile His Arg Trp Val Leu Ser Val Ile Asn Pro
225 230 235 240
Ser Glu Glu Asp Asn Gly Leu Leu Thr Ser Arg Ile Asn Leu His Cys
245 250 255
Lys Ala Ile Ile Ala Asp Tyr Ala Ser Lys Lys Gly Asp Val Lys Pro
260 265 270
Asn Ile Ile His Arg Met Leu Asp Ala Pro Glu Leu Ser Met Lys Asp
275 280 285
Lys Thr Ala Trp Arg Leu Gln Leu Glu Ala Arg Thr Leu Ile Gly Ala
290 295 300
Gly Thr Glu Thr Thr Gly His Thr Leu Ala Val Ile Ala Phe His Leu
305 310 315 320
Leu Ala Asn Pro Glu Lys Ala Lys Arg Leu Lys Glu Glu Ile Leu Ala
325 330 335
Thr Lys Glu Gly Arg Glu Lys Pro Leu Thr Tyr Gln Glu Leu Gln Met
340 345 350
Leu Pro Tyr Leu Ser Ser Val Val Leu Glu Gly His Arg Ile Ser Ser
355 360 365
Val Val Ser Gly Arg Leu Pro Arg Val Asn Thr Lys Glu Pro Leu Arg
370 375 380
Tyr Gly Asp Tyr Ser Ile Pro Ile Gly Thr Pro Val Ser Thr Thr Gln
385 390 395 400
Arg Leu Thr His Tyr Asn Ala Thr Ile Phe Pro Ser Pro Asn Thr Phe
405 410 415
Leu Pro Glu Arg Trp Leu Gln Pro Ser Glu Arg Lys Arg Leu Glu Lys
420 425 430
Tyr Ile Gln Pro Phe Gly Arg Gly Ser Arg Ser Cys Ile Gly Met His
435 440 445
Leu Ala Asn Ala Glu Ile Tyr Lys Thr Leu Ala Glu Met Phe Ala Arg
450 455 460
Phe Asp Met Lys Leu Tyr Asp Thr Glu Phe Glu Asp Ile Met Gln Val
465 470 475 480
His Asp Phe Phe Thr Ser Phe Pro Ser Ser Glu Arg Gly Leu Arg Ile
485 490 495
Leu Val Glu Ala
500
<210> 3
<211> 849
<212> DNA
<213> Artificial Sequence
<220>
<223> mcfS
<400> 3
atggctttag accgccagaa tgcgaaagtt acaactttcg gtctgtcaaa gccgaaaacc 60
aatatagatc gccgatcatg tcagagaact gtccccatga aggttctctg cctaggacta 120
tgtcgaaccg gcacttcctc attgcgtgcg gctctctttg agcttggcct tgatgatgtc 180
tatcacatgt gtagtgtgac ggaagagaat cccctcgact ccaagttgtg gaaagaggcc 240
ttcgacgcga aatatgaagg gatcggcaag ccctacggaa gagctgaatt tgacgcactc 300
ttgggtcatt gcatggcaac ctcggatttc cccagcgttg ccttcgctcc agaactcatc 360
gccgcttacc ccgaggcaaa gataattctc actgtacgag ataacgccga tgtctggtat 420
gactccgttc tcaacacgat ctggagagtc tccaacttcc ttcgcgctcc tccgagaact 480
ttaacccaac gagtcgttca agcgattctt cccaagccgg atttcaacat attcaagtac 540
agcccccttg gcaactttcc tgaggaaggc tgtcagtggt atagtgactg gaatgaagag 600
attagaactc tagccaaagg gagggacttc ttggaattca atgtaaagga gggatggggt 660
ccactctgta gattcttgga ggtggagcag ccggagacgc catttccaag agtcaatgat 720
tcaaatacat tcaaggaatt tcatgataag ggtttggagc aggatattca aagactggta 780
ggcataagta ctaagcttgt cgccgctgtt ggtgtattgg gtttggctgt ttggctagct 840
aggaagtag 849
<210> 4
<211> 282
<212> PRT
<213> Artificial Sequence
<220>
<223> mcfS
<400> 4
Met Ala Leu Asp Arg Gln Asn Ala Lys Val Thr Thr Phe Gly Leu Ser
1 5 10 15
Lys Pro Lys Thr Asn Ile Asp Arg Arg Ser Cys Gln Arg Thr Val Pro
20 25 30
Met Lys Val Leu Cys Leu Gly Leu Cys Arg Thr Gly Thr Ser Ser Leu
35 40 45
Arg Ala Ala Leu Phe Glu Leu Gly Leu Asp Asp Val Tyr His Met Cys
50 55 60
Ser Val Thr Glu Glu Asn Pro Leu Asp Ser Lys Leu Trp Lys Glu Ala
65 70 75 80
Phe Asp Ala Lys Tyr Glu Gly Ile Gly Lys Pro Tyr Gly Arg Ala Glu
85 90 95
Phe Asp Ala Leu Leu Gly His Cys Met Ala Thr Ser Asp Phe Pro Ser
100 105 110
Val Ala Phe Ala Pro Glu Leu Ile Ala Ala Tyr Pro Glu Ala Lys Ile
115 120 125
Ile Leu Thr Val Arg Asp Asn Ala Asp Val Trp Tyr Asp Ser Val Leu
130 135 140
Asn Thr Ile Trp Arg Val Ser Asn Phe Leu Arg Ala Pro Pro Arg Thr
145 150 155 160
Leu Thr Gln Arg Val Val Gln Ala Ile Leu Pro Lys Pro Asp Phe Asn
165 170 175
Ile Phe Lys Tyr Ser Pro Leu Gly Asn Phe Pro Glu Glu Gly Cys Gln
180 185 190
Trp Tyr Ser Asp Trp Asn Glu Glu Ile Arg Thr Leu Ala Lys Gly Arg
195 200 205
Asp Phe Leu Glu Phe Asn Val Lys Glu Gly Trp Gly Pro Leu Cys Arg
210 215 220
Phe Leu Glu Val Glu Gln Pro Glu Thr Pro Phe Pro Arg Val Asn Asp
225 230 235 240
Ser Asn Thr Phe Lys Glu Phe His Asp Lys Gly Leu Glu Gln Asp Ile
245 250 255
Gln Arg Leu Val Gly Ile Ser Thr Lys Leu Val Ala Ala Val Gly Val
260 265 270
Leu Gly Leu Ala Val Trp Leu Ala Arg Lys
275 280

Claims (10)

1. A genetically engineered strain for high-yield micafungin precursor FR901379, wherein the genetically engineered strain is a genetically engineered strain for over-expressing cytochrome P450 monooxygenase and/or sulfonyl transferase, and the original strain of the genetically engineered strain is a phoma fungus;
the cytochrome P450 monooxygenase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 2;
the sulfotransferase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity compared to SEQ ID No. 4.
2. Genetically engineered strain according to claim 1, wherein the cytochrome P450 monooxygenase and/or sulfonyltransferase is derived from a phoma fungus.
3. A method of preparing/producing micafungin precursor FR901379, comprising the step of culturing the genetically engineered strain of claim 1 or 2.
4. A method according to claim 3, characterized in that the method further comprises the step of isolating/purifying FR 901379.
5. Use of a genetically engineered strain according to claim 1 or 2 for the preparation/production of micafungin precursor FR 901379.
6. A method of preparing the genetically engineered strain of claim 1, the method comprising the step of overexpressing the cytochrome P450 monooxygenase and/or sulfonyltransferase in a starting strain.
7. Use of a cytochrome P450 monooxygenase and/or a sulfonyltransferase as described in claim 1 or 2, or of a gene encoding it, for the preparation of a genetically engineered strain of high-yielding micafungin precursor FR901379, the starting strain of which is a fungus of the genus phoma.
8. Use of a cytochrome P450 monooxygenase and/or a sulfonyltransferase as described in claim 1 or 2 or of a biological material comprising a cytochrome P450 monooxygenase and/or a sulfonyltransferase as described in claim 1 for the preparation of micafungin precursor FR 901379.
9. The use according to claim 8, wherein the biological material is selected from the group consisting of: a vector comprising the cytochrome P450 monooxygenase and/or sulfonyltransferase of claim 1 or 2, or a gene encoding the same; alternatively, a host cell comprising the cytochrome P450 monooxygenase and/or sulfonyltransferase of claim 1 or 2, or a gene encoding the same.
10. Use of a gene encoding a cytochrome P450 monooxygenase and/or a sulfonyltransferase as described in claim 1 or 2 for the preparation of micafungin precursor FR 901379.
CN202210570328.1A 2022-05-24 2022-05-24 Strain with high yield of FR901379 and construction method and application thereof Pending CN117143748A (en)

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