CN117143749A - Strain for high-yield FR901379 and construction method and application thereof - Google Patents

Strain for high-yield FR901379 and construction method and application thereof Download PDF

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CN117143749A
CN117143749A CN202210570355.9A CN202210570355A CN117143749A CN 117143749 A CN117143749 A CN 117143749A CN 202210570355 A CN202210570355 A CN 202210570355A CN 117143749 A CN117143749 A CN 117143749A
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monooxygenase
cytochrome
strain
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吕雪峰
门萍
黄雪年
周宇
谢丽
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 an original strain of the genetic engineering strain is a phoma fungus.

Description

Strain for high-yield 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 can improve the yield of FR901379 by over-expressing a target gene, and a construction method and application thereof.
Background
Echinocandin antifungal drugs are derivatives of a class of cyclic lipopeptides natural products, and can selectively inhibit the activity of beta-1, 3 glucan synthase in fungal cell walls, thereby affecting the synthesis of the fungal cell walls and leading to the lysis and death of the fungal cells. The echinocandins medicine has unique action mechanism, high safety, wide antibacterial spectrum and is effective on drug-resistant bacteria. With the aggravation of fungal resistance, the proportion of echinocandins in the global antifungal drug market is in an increasing trend year by year. Currently, clinically applied echinocandin antifungal drugs are caspofungin, micafungin and anidulafungin. Among them, micafungin has its uniqueness, and its precursor FR901379 has a sulfonyl group, thereby having excellent water solubility, and thus improving its bioavailability.
The industrial production of micafungin comprises three steps: firstly, FR901379 is produced by Coleophoma empetri fermentation, then fatty acid side chains are hydrolyzed by fermentation of S.natalii, and finally, micafungin is finally produced by adding 4- (5- (4- (pentyloxy) phenyl) -3-isoxazolyl) methyl benzoate side chains through chemical modification. Wherein, the fermentation of C.empetri to produce FR901379 is an important step in the production process and is important for the control of the production cost and the post-treatment process. At present, the optimization of the production performance of the C.empetri is generally performed by means of fermentation medium optimization, physical mutagenesis and the like, and no relevant report on improving the production performance of the strain through genetic modification exists. In addition to FR901379, two other byproducts WF11899B and WF11899C exist in the fermentation product of the EMpetri, and the biggest difference between the byproducts and the FR901379 is that the hydroxylation modification degree of the C-4 position of the L-ornithine is different from that of the C-4 position of the L-homotyrosine. The phenomenon of low yield and more byproducts exists in the fermentation process of C.empetri, so that the subsequent separation and purification are difficult, and the production cost of micafungin is always high. The industrial production of micafungin is now in urgent need of solving the above problems, but the coding genes of enzymes responsible for hydroxylation of the C-4 position of L-ornithine and the C-4 position of L-homotyrosine are not known, which greatly limits the related metabolic engineering. Therefore, the identification of oxidase responsible for hydroxylation of the L-ornithine C-4 site and the L-homotyrosine C-4 site provides an improvement target point for metabolic engineering, reduces byproduct accumulation, improves the yield of FR901379, and promotes the quality improvement and the efficiency of the micafungin production technology.
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 the invention, different genetic engineering strains are obtained aiming at cytochrome P450 monooxygenase McfF and McfH in the Phoma sphaerocarpum (Coleophoma sp.) MEFC009, and certain genetic engineering strains can produce micafungin precursor FR901379.
In the invention, mcfF 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 McfH is cytochrome P450 monooxygenase (cytochrome P450 monooxygenase), the amino acid sequence of the McfH is shown as SEQ ID No.4, and the nucleic acid sequence of the McfH 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% or 99% sequence identity compared to SEQ ID No.2 or 4; 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%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No.2 or 4, and the cytochrome P450 monooxygenase is derived from a fungus of the genus phophoma. The phoma fungi include Coleophoma sp.or Coleophoma empetri, for example, coleophoma sp.sphaeroides MEFC009.
In a preferred embodiment, the amino acid sequence of the cytochrome P450 monooxygenase is shown in SEQ ID No.2 or SEQ ID No. 4.
In another aspect, the present invention also provides a biomaterial comprising the cytochrome P450 monooxygenase 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 a host cell comprising the above cytochrome P450 monooxygenase.
In another aspect, the invention also provides a gene encoding the cytochrome P450 monooxygenase 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, pUolADuet-1, pRSFDuet-1, plP-OmpA, pUC-series vectors (e.g., pUC18, pUC 19), pQE-30, pXH-1, pPtd43, and XHdRII.
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), top 10, JM 109), yeast (e.g., saccharomyces cerevisiae, pichia pastoris, yarrowia lipolytica), and Phoma sheath.
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.
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.
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.
The genetic engineering strain for preparing the high-yield micafungin precursor FR901379 is prepared by introducing the cytochrome P450 monooxygenase 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 can 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):
in the invention, structural formulas of WF11899B and WF11899C are respectively shown as formula (II) and formula (III):
the invention also provides a compound, the structural formula of which is shown as the formula (IV):
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 an original strain of the genetic engineering bacterium is a phoma sphaeroides fungus.
In one embodiment, the first cytochrome P450 monooxygenase is overexpressed in the genetically engineered bacterium; the first cytochrome P450 monooxygenase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity compared to SEQ ID No. 2; preferably, the first cytochrome P450 monooxygenase is derived from a fungus of the genus phophoma; more preferably, the amino acid sequence of the first cytochrome P450 monooxygenase has at least 70% sequence identity compared to SEQ ID No.2, and the first cytochrome P450 monooxygenase is derived from a fungus of the genus phophoma.
In one embodiment, the second cytochrome P450 monooxygenase is overexpressed in the genetically engineered bacterium; the second cytochrome P450 monooxygenase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity compared to SEQ ID No. 4; preferably, the second cytochrome P450 monooxygenase is derived from a fungus of the genus phophophoma; more preferably, the amino acid sequence of the second cytochrome P450 monooxygenase has at least 70% sequence identity compared to SEQ ID No.4, and the second cytochrome P450 monooxygenase is derived from a fungus of the genus phophoma.
In one embodiment, the first cytochrome P450 monooxygenase and the second cytochrome P450 monooxygenase are overexpressed simultaneously 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 genetic engineering strain for high-yield WF11899B, wherein the WF11899B is shown in the formula II, and the original strain of the genetic engineering strain is a phoma sphaeroides fungus. Further, the genetically engineered strain of the high-yield WF11899B is a genetically engineered strain obtained by mutating cytochrome P450 monooxygenase in a starting strain; 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.
In another aspect, the present invention also provides a method for preparing/producing WF11899B 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 WF 11899B.
In another aspect, the present invention also provides a method for preparing/producing WF11899B, which comprises the step of culturing using the genetically engineered strain; preferably, the method further comprises the step of isolating/purifying WF 11899B.
On the other hand, the invention also provides a genetically engineered strain for high-yield of the compound shown in the formula IV, wherein the original strain of the genetically engineered strain is the phoma sphaeroides fungus. Furthermore, the genetically engineered strain of the high-yield compound shown in the formula IV is a genetically engineered strain obtained by mutating cytochrome P450 monooxygenase in a starting strain; the cytochrome P450 monooxygenase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 4.
In another aspect, the present invention also provides a method for preparing/producing a compound represented by formula IV 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 the compound shown in the formula IV.
In another aspect, the present invention also provides a method for preparing/producing a compound represented by formula IV, comprising the step of culturing using the above genetically engineered strain; preferably, the method further comprises the step of isolating/purifying the compound of formula IV.
The mutation described in the present invention includes a loss of gene function or activity by means of gene deletion, gene insertion or gene substitution.
In a preferred embodiment, the gene mutation is a gene knockout of the target gene.
In one embodiment, the gene mutation may be accomplished using techniques conventional in the art, for example, by homologous recombination to knock-in or knock-out resulting in loss of gene function or activity; alternatively, the gene may be mutated using gene editing means, such as zinc finger endonuclease (ZFN), transcription activator-like effector nuclease (TALEN) or CRIspR techniques, resulting in loss of gene function or activity.
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 mcfF gene; wherein # 2, # 3 and # 5 are transformants deleted of the gene mcfF, and WT is the control strain Coleophoma sp.mefc009.
FIG. 2 is the results of HPLC analysis of the fermentation product of the gene mcfF deleted strain Coleophoma sp. - Δmcff; wherein Coleophoma sp. - Δmcff is a gene mcfF deleted strain, and WT is a control strain Coleophoma sp.mefc009;1: FR901379;2: WF11899B;3: WF11899C.
FIG. 3 is the chemical structures of compounds FR901379, WF11899B and WF 11899C;
FIG. 4 is a result of genome PCR verification of transformants obtained by knocking out mcfH gene; wherein 6# and 8# are transformants with the mcfH gene deleted, and WT is the control strain Coleophoma sp.mefc009.
FIG. 5 is the HPLC analysis result of the gene mcfH deletion strain Coleophoma sp.—Δmcfh fermentation product; wherein Coleophoma sp. - Δmcfh is a gene mcfH deleted strain, and WT is a control strain Coleophoma sp.mefc009;1: FR901379;2: WF11899B;3: WF11899C.
FIG. 6 is the results of LC-MS analysis of compound 4 in the fermentation product of the gene mcfH deleted strain Coleophoma sp.
FIG. 7 is a chemical structure of Compound 4 in a fermentation product of a Gene mcfH deleted strain Coleophoma sp. - Δmcfh;
FIG. 8 is a genomic PCR validation of transformants obtained over-expressing the P450 enzyme McfF; wherein 1-23 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 McfF; wherein 1: FR901379;2: WF11899B;3: WF11899C.
FIG. 10 is an analysis of the yield of FR901379 (WF 11899A), WF11899B and WF11899C in engineering strains and Coleophoma sp.MEFC009 fermentation broth over-expressing the P450 enzyme McfF;
FIG. 11 is a genomic PCR validation of the transformant obtained by over-expression of the P450 enzyme McfH; wherein 1-23 are transformants and WT is wild-type Coleophoma sp.MEFC009.
FIG. 12 shows the HPLC analysis results of engineering strains and Coleophoma sp.MEFC009 fermentation products over-expressing the P450 enzyme McfH; wherein 1: FR901379;2: WF11899B;3: WF11899C.
FIG. 13 is an analysis of the yield of FR901379 (WF 11899A), WF11899B and WF11899C in engineering strains and Coleophoma sp.MEFC009 fermentation broth over-expressing the P450 enzyme McfH.
FIG. 14 is a genomic PCR validation of transformants obtained by simultaneously overexpressing the P450 enzymes McfF and McfH; wherein 1-18 are transformants and WT is wild-type Coleophoma sp.MEFC009.
FIG. 15 is an analysis of the yield of FR901379 (WF 11899A, WF11899B and WF 11899C) in engineering strains and Coleophoma sp.MEFC009 fermentation broth over-expressing the P450 enzymes McfF and McfH simultaneously.
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 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 enzymes Ultra 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 。
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 。
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 plate 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.
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.
Example 1 construction of an engineering strain with knocked out Gene mcfF Coleophoma sp.—DeltamcfF
PCR amplification was performed using the genome of wild-type Coleophoma sp.MEFC009 as a template, pfu DNA polymerase (Fermentas, catalog No.: EP 0501), primers UmcfF-F (5'-cacctctaagatagtctatc-3') and UmcfF-R (5'-ctttacgcttgcgatcccgaaTGTATAAGATGCATCAGTGCC-3') were used to amplify an upstream sequence U-mcfF of approximately 1.2kb in size, and primers DmcfF-F (5'-cctgggttcgcaaagataattgCTCGAACGTTGGATATATAGC-3') and DmcfF-R (5'-ttgccaaaacaggctctgata-3') were used to amplify a downstream sequence D-mcfF of 1.2kb in size. 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-mcfF and the downstream sequence D-mcfF are fused by fusion PCR, and then nest primers UmcfF-CS-F (5'-ggatactttcaattatgcggcc-3') and DmcfF-CS-R (5'-aattgagggacagtcattct-3') are used for amplifying a knockout targeting element UmcfF-hph-DmcfF with the size of 4.4kb by PCR.
Taking Coleophoma sp.MEFC009 as a starting strain, firstly taking a small amount of mycelium from a PDA 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 into 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. The mycelium is washed to white, 1g 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 and 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 UmcfF-hph-DmcfF fragment was added, followed by 50. Mu.l of PSTC, gently mixed and ice-cooled for 30min. Adding 1ml of PSTC, uniformly mixing, and standing at room temperature for 20min; then, the mixture was mixed with 10ml of top agar and poured onto 3 regeneration screening media plates PDA-SH, and cultured at 30℃in the dark for 5-7 days, 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 successive passages for 3 passages. Selecting 3 transformants 2# and 3# and 5# of stable passage, performing monospore separation and purification, and extracting the genome of the transformant after monospore separation. PCR verification of the transformant genome using the external primers UmcfF-F (5'-cacctctaagatagtctatc-3') and DmcfF-R (5'-ttgccaaaacaggctctgata-3') allowed the amplification of a band of about 4.7kb as positive transformants, whereas Coleophoma sp.MEFC009 was able to amplify only a band of about 3.2kb, and the results in FIG. 1 indicate that the 2#,3#,5# transformants were positive transformants, indicating homologous recombination at the mcfF position of the gene, incorporating the exogenous fragment UmcfF-hph-DmcfF, defining the positive strain as Coleophoma sp.
Example 2 fermentation verification of Gene mcfF deletion engineering Strain Coleophoma sp.—ΔmcfF
3 positive engineering strains No.2, no. 3, no. 5 and control strains Coleophoma sp.MEFC009 were inoculated on PDA solid plates and cultured 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 (250 ml triangular flask) of Coleophoma sp. At 25 ℃,220rpm, shaking culture for 45-48h. The seed solution from the above culture was taken in 5ml to 50ml of Coleophoma sp. Fermentation medium (250 ml Erlenmeyer flask), 25℃and 220rpm, and shake-cultured for 8 days, with 3 replicates per strain. 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 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; compound 1 (FR 901379) in Coleophoma sp.—Δmcff disappeared compared to the starting strain, the yield of the corresponding compound 2 (WF 11899B) was the sum of compound 1 and compound 2 in the starting strain Coleophoma sp..and compound WF11899B lacks a hydroxyl group at the C-4 position of L-homotyrosine compared to FR901379 (as shown in FIG. 3), indicating that the P450 enzyme encoded by the gene mcfF responsible for hydroxylation at the C-4 position of L-homotyrosine, the nucleic acid sequence of which is shown in SEQ ID No.1, and the amino acid sequence encoded by which is shown in SEQ ID No. 2.
Example 3 construction of an engineering strain with knocked out Gene mcfH, coleophoma sp. - Δmcfh
PCR amplification was performed using the genome of wild-type Coleophoma sp.MEFC009 as a template, pfu DNA polymerase (Fermentas, catalog No.: EP 0501), primers UmcfH-F (5'-gtgagtgttcctcaaggcag-3') and UmcfH-R (5'-ctttacgcttgcgatcccgaaATCACCGATCAGACCATCTC-3') were used to amplify an upstream sequence U-mcfH of approximately 1.5kb in size, and primers DmcfH-F (5'-cctgggttcgcaaagataattgCGCCAAGTTGTCAGCCCAAA-3') and DmcfH-R (5'-ccgctttaatcaacttggca-3') were used to amplify a downstream sequence D-mcfH of 1.4kb in size. 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-mcfH and the downstream sequence D-mcfH are fused by fusion PCR, and then nest primers Umcfh-CS-F (5'-atagcctattcatgatttct-3') and Dmcfh-CS-R (5'-tacgcccgagcgacccgagt-3') are used for amplifying a knockout targeting element Umcfh-hph-Dmcfh with a size of 4.8kb by PCR by taking the fusion product as a template.
Taking Coleophoma sp.MEFC009 as a starting strain, firstly taking a small amount of mycelium from a PDA 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 into 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. The mycelium is washed to white, 1g 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% snailaseAnd 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 UmcfH-hph-DmcfH fragment was added, followed by 50. Mu.l of PSTC, gently mixed and ice-cooled for 30min. Adding 1ml of PSTC, uniformly mixing, and standing at room temperature for 20min; then, the mixture was mixed with 10ml of top agar and poured onto 3 regeneration screening media plates PDA-SH, and cultured at 30℃in the dark for 5-7 days, 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 successive passages for 3 passages. Selecting 3 transformants 6# and 8# and 9# of 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 Umcfh-F (5'-gtgagtgttcctcaaggcag-3') and Dmcfh-R (5'-ccgctttaatcaacttggca-3') allowed the amplification of a band of approximately 5.0kb as positive transformants, whereas Coleophoma sp.MEFC009 was only able to amplify a band of approximately 3.7kb, FIG. 4 illustrates that at 6#,8#,9# transformants were positive transformants, indicating that homologous recombination occurred at the position of the gene mcfH, integrating the exogenous fragment Umcfh-hph-Dmcfh, defining the positive strain as Coleophoma sp. - Δmcfh.
Example 4 fermentation verification of Gene mcfH deletion engineering Strain Coleophoma sp.—Δmcfh
3 engineering strains 6# and 8# and 9# and a control strain Coleophoma sp.MEFC009 were inoculated on a PDA solid plate and cultured 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) of Coleophoma sp. At 25 ℃,220rpm, shake cultivation45-48h. The seed solution from the above culture was taken in 5ml to 50ml of Coleophoma sp. Fermentation medium (250 ml Erlenmeyer flask), 25℃and 220rpm, and shake-cultured for 8 days, with 3 replicates per strain. 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 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. 5; coleophoma sp. - Δmcfh disappeared from the original strain Coleophoma sp.MEFC009, and compound 4 appeared accordingly.
Compound 4 was isolated and purified by semi-preparative liquid chromatography (HITACHI print). The preparation method comprises the following steps: 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 2ml/min, ultraviolet detection wavelength: 210nm,30 ℃; the elution was performed isocratically with mobile phase B accounting for 55% of the mobile phase volume and total elution time of 20min. And compound 4 was analyzed by liquid chromatography-mass spectrometry (LC-MS) and Nuclear Magnetic Resonance (NMR). 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; the elution conditions were: gradient elution conditions: 0-1min, mobile phase B linearly increasing from 5% to 20% by volume, 1-6min, mobile phase B linearly increasing from 20% to 60% by volume, 6-7min, mobile phase B occupying the volume of mobile phaseLinearly from 60% to 100%. As shown in FIG. 6, the mass-to-charge ratio [ M+H ] of Compound 4 ] + 1127.5542 (C) 51 H 82 N 8 O 18 S, theoretical value: 1127.5541). The structure of compound 4 is shown in FIG. 7 in combination with the NMR analysis results. In comparison with FR901379, compound 4 lacks 3 hydroxyl groups at both the C-4 and C-5 positions of L-homotyrosine, and the P450 enzyme McfF is known to be responsible for hydroxylation of the C-4 position of L-homotyrosine, so the P450 enzyme McfH is known to be responsible for hydroxylation of the C-4 and C-5 positions of L-ornithine, and affects the effect of the gene mcfF when the gene mcfH is knocked out. The nucleic acid sequence of mcfH is shown as SEQ ID No.3, and the encoded amino acid sequence is shown as SEQ ID No. 4.
EXAMPLE 5 construction of recombinant strains overexpressing the P450 enzyme McfF
5.1 construction of the expression cassette for the overexpression of the P450 enzyme McfF
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 was performed using the genome of Coleophoma sp.MEFC009 as a template and using primers mcfF-F (5'-caactcatcaatcatcacaacATGCTTTCAGACACGACGGC-3') and mcfF-R (5'-gatttcagtaacgttaagtggCTATTCCGTCCGCCTTCTTA-3') to obtain a fragment mcfF of about 1.7kb in size; using plasmid pXH-1 as template and using primers TtrPC-F (5'-ccacttaacgttactgaaat-3') and TtrPC-R (5'-tacctctaaacaagtgtacc-3') as primers to make PCR amplification so as to obtain terminator fragment TtrPC whose size is about 0.7 kb; fusion PCR is carried out on the fragments PgpdAT, mcfF and TtrPC, so that the expression cassette PgpdAT-mcfF-TtrPC 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 McfF
Taking Coleophoma sp.MEFC009 as starting strain, firstly taking a small amount of mycelium from PDA plate, crushing with a hand-held homogenizer, and collecting 1ml seedThe seed solution was inoculated into 50ml of seed medium, and shake culture was performed at 25℃in a 250ml Erlenmeyer flask at 220 rpm. 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 into 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. The mycelium is washed to white, 1g 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 and 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, pgpdAT-mcfF-TtrpC 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, the mixture was mixed with 10ml of top agar and poured onto 3 regeneration screening media plates PDA-SH, and cultured at 30℃in the dark for 5-7 days, 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 23 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 Ttrpc-R (5'-tacctctaaacaagtgtacc-3'), wherein the positive transformants can be amplified by a band with a size of about 3.2kb, as shown in FIG. 8; of the 23 transformants, the other transformants were positive transformants except for transformant 21#, and the expression element PgpdAT-mcfF-TtrPC was integrated on the genome.
Example 6 fermentation verification of recombinant strains overexpressing the P450 enzyme McfF
Will be solidThe engineered strain overexpressing mcfF 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 seed culture medium of Coleophoma sp (250 ml triangular flask), 25 ℃,220rpm, shaking culture for 45-48h. The above cultured seed solution was shake-cultured at 25℃and 220rpm for 8 days with 5ml of a 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 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. As shown in FIG. 9, the results of HPLC analysis revealed that the yield of compound FR901379 in mcfF was increased and the yield of by-product WF11899B was decreased as compared with the wild-type Coleophoma sp.MEFC009. As shown in FIG. 10, the yield of FR901379 in the engineering strain over-expressing mcfF is higher than that of the control strain Coleophoma sp.MEFC009, and the yield of FR901379 in the engineering strain over-expressing mcfF is 706mg/L, which is 130% higher than that of the control strain Coleophoma sp.MEFC009. In addition to the Phoma sphaeroides (Coleophoma sp.) MEFC009, strain Coleophoma empetri F-11899 can also produce FR901379. We also overexpressed the mcfF homologous gene BAN91490.1 in C.empetriF-11899 (Sequence ID in NCBI), and the yield of FR901379 was increased by 100% compared to the control strain C.empetriF-11899.
EXAMPLE 7 construction of recombinant strains overexpressing the P450 enzyme McfH
PCR amplification was performed using the genome of Coleophoma sp.MEFC009 as a template and the primers mcfH-F (5'-caactcatcaatcatcacaacATGGTTCCATCAATGATCTC-3') and mcfH-R (5'-gatttcagtaacgttaagtggTCACAGGGCTACTTTCGATC-3') to obtain a fragment mcfH of about 1.7kb in size; the fragment PgpdAT, ttrpC and the fragment mcfH in example 5 were fused by fusion PCR to obtain the expression cassette PgpdAT-mcfH-TtrpC.
Taking Coleophoma sp.MEFC009 as a starting strain, firstly taking a small amount of mycelium from a PDA 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 into 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. The mycelium is washed to white, 1g 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 and 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, pgpdAT-mcfH-TtrpC 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, the mixture was mixed with 10ml of top agar and poured onto 3 regeneration screening media plates PDA-SH, and cultured at 30℃in the dark for 5-7 days, 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 23 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 Ttrpc-R (5'-tacctctaaacaagtgtacc-3'), wherein the positive transformants can be amplified by a band with a size of about 3.2kb, as shown in FIG. 11; all of these 23 transformants were positive transformants, and the expression element PgpdAT-mcfH-TtrPC was integrated on the genome.
Example 8 fermentation verification of recombinant strains overexpressing the P450 enzyme McfH
The engineering strain and the control strain Coleophoma sp.MEFC009, which overexpress the P450 enzyme McfH in example 7, 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 (250 ml triangular flask) of Coleophoma sp. At 25 ℃,220rpm, shaking culture for 45-48h. The above cultured seed solution was shake-cultured at 25℃and 220rpm for 8 days with 5ml of a 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 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. As shown in FIG. 12, the results of HPLC analysis revealed that the yield of compound FR901379 in mcfH was increased, and the accumulation of by-product WF11899C was reduced and hardly produced, compared with the wild-type Coleophoma sp.MEFC009. As shown in FIG. 13, the yield of FR901379 in the engineering strain over-expressing mcfH was higher than that of the control strain Coleophoma sp.MEFC009, and the yield of FR901379 in the engineering strain over-expressing mcfH was 436mg/L, which was 44% higher than that of the control strain Coleophoma sp.MEFC009. We also overexpressed the mcfH homologous gene BAN91489.1 in C.empetriF-11899 (Sequence ID in NCBI), and the yield of FR901379 was increased by 32.8% compared to the control strain C.empetriF-11899.
EXAMPLE 9 construction of recombinant strains that overexpress the P450 enzymes McfF and McfH simultaneously
Taking Coleophoma sp.MEFC009 as a starting strain, firstly taking a small amount of mycelium from a PDA 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 into 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. The mycelium is washed to white, 1g 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 and 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.
The expression cassettes PgpdAT-mcfF-TtrPC, pgpdAT-mcfH-TtrPC and hph constructed in the previous examples 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, the mixture was mixed with 10ml of top agar and poured onto 3 regeneration screening media plates PDA-SH, and cultured at 30℃in the dark for 5-7 days, 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 18 transformants for separation and purification, and performing PCR (polymerase chain reaction) verification on the purified transformant gene groups by using primers PgpdAT-F (5'-ccctgggttcgcaaagataattggttacactctgggaggatcc-3') and mcfF-R (5'-gatttcagtaacgttaagtggCTATTCCGTCCGCCTTCTTA-3'), wherein PgpdAT-F (5'-ccctgggttcgcaaagataattggttacactctgggaggatcc-3') and mcfH-R (5'-gatttcagtaacgttaagtggTCACAGGGCTACTTTCGATC-3') can simultaneously amplify a band with the size of about 2.5kb as a positive transformant, as shown in FIG. 14; the 18 transformants are positive transformants except for the transformants 1#,16#,17#, and the expression elements PgpdAT-mcfF-TtrPC and PgpdAT-mcfH-TtrPC are integrated on the genome.
Example 10 fermentation verification of recombinant strains simultaneously overexpressing the P450 enzymes McfF and McfH
Recombinant strain and control strain, coleophoma sp.MEFC009, which overexpress the P450 enzymes McfF and McfH simultaneously 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 to 50ml of seed culture medium (250 ml triangular flask) of Coleophoma sp. At 25 ℃,220rpm, shaking culture for 45-48h. The seed solution from the above culture was taken in 5ml to 50ml of Coleophoma sp. Fermentation medium (250 ml Erlenmeyer flask), 25℃and 220rpm, and shake-cultured for 8 days, with 3 replicates per strain. 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 fermentation results are shown in FIG. 15, and the engineering strain Coleophoma sp over-expressing mcfF and mcfH simultaneously has the advantages that the yield of FR901379 is improved, and two byproducts WF11899B and WF11899C are accumulated compared with the control strainWhile decreasing. We also overexpressed the homologous genes BAN91490.1, BAN91489.1 (Sequence ID in NCBI) for mcfF and mcfH in C.empetrif-11899, with increased yield of FR901379 and decreased accumulation of both byproducts WF11899B and WF11899C compared to control strain C.empetrif-11899.
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> a strain with high FR901379 yield, construction method and application thereof
<130> 111
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1641
<212> DNA
<213> Artificial Sequence
<220>
<223> mcfF
<400> 1
atgctttcag acacgacggc tcgcatagag cgcatcatta gcgaacagac tttattcagc 60
gctgtactca gtttgtttat gataggcctc atggctcatc tcgtgcttgc acgcttctcg 120
atacataacc aattctggag tgctcaagta tggacaggag ttcgtgcaga atggtttccc 180
aagataagag caaaattccg caccattggc ggtatacgcc aaatgttaag cgatggctat 240
aaatgctttt caaaacagga gagagcattt gttttgccca tgctcggcga gaaaccgtgg 300
ctcgtgctac ctccttccag cattcctgag cttcttgcaa agtctgattc agaggttgat 360
atgcgcatag tccacgagca acagctgcaa catgagtata cgcaaggcgc cctcggtcgc 420
catgttgtcg acgtgcccat tcaatacgat gttatccatc gtcaattgaa tcgaaagctt 480
cctcatttga tagacccctt taatgatgag tttgataaaa gcttcagaaa atactggggc 540
actgatgcat cttatacaga tgtaaaagtc tcagcaacat gcgaaaagat aatcgctcaa 600
gttgcaaacc gaattttcgc aggcccagaa atctgtcgga atgaggattt cttggaacac 660
tctaggctct attcagcagg agtagggagg tgtgcaatca tcctacgtat gcttccacag 720
gtaatacgat cattagttgc accgcttgtt acatacccaa accgcaaaca ccacgatgtt 780
tgcttgagag tctgcctccc agttgtaaga gacaggctac aacgaacctc tgagaagaga 840
ggcaatcttc aatctgagtg ggaaccgccg gtggatatgc ttcaatggat tatcgaagaa 900
gcttttaatc gcaatgaacc aaaagagctt gatgctcacc tgatcactca acggatactt 960
aagcttaact ttgtctccat cgaaactatt cacatgtcta tgacccatgc cattctcgat 1020
ctttaccgct cacctcattc cgagagattt gtagctggcc ttcgtcaaga atgtgatcgc 1080
gtacttgaag cgaataatgg ccaatggacc aagagcgggc tcgatgacct cttgtgcatt 1140
gactcgacaa tccgcgagtc gatgagatac tcgaacgttg gatatatagc actgactcga 1200
atggttgtcg acccgcatgg gacccaattt catgctaatg gcaaaggcaa cagcagtcca 1260
atgtccatac cttctggcat ccgagtctgc gtgcccgccc atgcaatcca cagagaccct 1320
gagttttatt cttccccaca tgaatttcaa gccttccgct ttgctgaagc ttatgaaaag 1380
aatagaaaca taggaaacga atcttacgag gccaagatat ctatcgtcac cacgacagat 1440
aaatttctgc ccttcggcca cggccgccac gcatgcccag gccgcttttt tgccgctcag 1500
atgatgaagc taatgctggt ttacttggtg caaaattacg acgtggaaaa gctatccaca 1560
ggagtagaaa acaaagtcac ggtagggacc gcgaagcctg acagtaattt gagtttaaga 1620
gtaagaaggc ggacggaata g 1641
<210> 2
<211> 546
<212> PRT
<213> Artificial Sequence
<220>
<223> McfF
<400> 2
Met Leu Ser Asp Thr Thr Ala Arg Ile Glu Arg Ile Ile Ser Glu Gln
1 5 10 15
Thr Leu Phe Ser Ala Val Leu Ser Leu Phe Met Ile Gly Leu Met Ala
20 25 30
His Leu Val Leu Ala Arg Phe Ser Ile His Asn Gln Phe Trp Ser Ala
35 40 45
Gln Val Trp Thr Gly Val Arg Ala Glu Trp Phe Pro Lys Ile Arg Ala
50 55 60
Lys Phe Arg Thr Ile Gly Gly Ile Arg Gln Met Leu Ser Asp Gly Tyr
65 70 75 80
Lys Cys Phe Ser Lys Gln Glu Arg Ala Phe Val Leu Pro Met Leu Gly
85 90 95
Glu Lys Pro Trp Leu Val Leu Pro Pro Ser Ser Ile Pro Glu Leu Leu
100 105 110
Ala Lys Ser Asp Ser Glu Val Asp Met Arg Ile Val His Glu Gln Gln
115 120 125
Leu Gln His Glu Tyr Thr Gln Gly Ala Leu Gly Arg His Val Val Asp
130 135 140
Val Pro Ile Gln Tyr Asp Val Ile His Arg Gln Leu Asn Arg Lys Leu
145 150 155 160
Pro His Leu Ile Asp Pro Phe Asn Asp Glu Phe Asp Lys Ser Phe Arg
165 170 175
Lys Tyr Trp Gly Thr Asp Ala Ser Tyr Thr Asp Val Lys Val Ser Ala
180 185 190
Thr Cys Glu Lys Ile Ile Ala Gln Val Ala Asn Arg Ile Phe Ala Gly
195 200 205
Pro Glu Ile Cys Arg Asn Glu Asp Phe Leu Glu His Ser Arg Leu Tyr
210 215 220
Ser Ala Gly Val Gly Arg Cys Ala Ile Ile Leu Arg Met Leu Pro Gln
225 230 235 240
Val Ile Arg Ser Leu Val Ala Pro Leu Val Thr Tyr Pro Asn Arg Lys
245 250 255
His His Asp Val Cys Leu Arg Val Cys Leu Pro Val Val Arg Asp Arg
260 265 270
Leu Gln Arg Thr Ser Glu Lys Arg Gly Asn Leu Gln Ser Glu Trp Glu
275 280 285
Pro Pro Val Asp Met Leu Gln Trp Ile Ile Glu Glu Ala Phe Asn Arg
290 295 300
Asn Glu Pro Lys Glu Leu Asp Ala His Leu Ile Thr Gln Arg Ile Leu
305 310 315 320
Lys Leu Asn Phe Val Ser Ile Glu Thr Ile His Met Ser Met Thr His
325 330 335
Ala Ile Leu Asp Leu Tyr Arg Ser Pro His Ser Glu Arg Phe Val Ala
340 345 350
Gly Leu Arg Gln Glu Cys Asp Arg Val Leu Glu Ala Asn Asn Gly Gln
355 360 365
Trp Thr Lys Ser Gly Leu Asp Asp Leu Leu Cys Ile Asp Ser Thr Ile
370 375 380
Arg Glu Ser Met Arg Tyr Ser Asn Val Gly Tyr Ile Ala Leu Thr Arg
385 390 395 400
Met Val Val Asp Pro His Gly Thr Gln Phe His Ala Asn Gly Lys Gly
405 410 415
Asn Ser Ser Pro Met Ser Ile Pro Ser Gly Ile Arg Val Cys Val Pro
420 425 430
Ala His Ala Ile His Arg Asp Pro Glu Phe Tyr Ser Ser Pro His Glu
435 440 445
Phe Gln Ala Phe Arg Phe Ala Glu Ala Tyr Glu Lys Asn Arg Asn Ile
450 455 460
Gly Asn Glu Ser Tyr Glu Ala Lys Ile Ser Ile Val Thr Thr Thr Asp
465 470 475 480
Lys Phe Leu Pro Phe Gly His Gly Arg His Ala Cys Pro Gly Arg Phe
485 490 495
Phe Ala Ala Gln Met Met Lys Leu Met Leu Val Tyr Leu Val Gln Asn
500 505 510
Tyr Asp Val Glu Lys Leu Ser Thr Gly Val Glu Asn Lys Val Thr Val
515 520 525
Gly Thr Ala Lys Pro Asp Ser Asn Leu Ser Leu Arg Val Arg Arg Arg
530 535 540
Thr Glu
545
<210> 3
<211> 1593
<212> DNA
<213> Artificial Sequence
<220>
<223> mcfH
<400> 3
atggttccat caatgatctc ggcagtggtc ccaataatca cctccacaaa tctggttttt 60
tatggagcaa ctggacttgt cctatttgct atcctcgcct actcacttaa cagattgaca 120
acttgggaat actcgatccc aaacgaagta caatggattg atcgtcgcaa agagtctttc 180
tcttatcttc gcgcaaaagc ccgatcgctt gttagaaaca aagaaaatgt gcttgaagct 240
tatttcaagt tcaacaaact tggtaatgca gcagcatgtg cggttgcttt cggtcgtcca 300
ttgttgcttc tgccgcctac tttcatccgc tggattgttg atcaacctga atctaccatc 360
agtttggatc cgatacacga tgacttccat gcatttgttg gagatggtct gatcggtgat 420
cataccgtgc aggagctgtt acgccgagag ttgactctta acctggacaa gttgacgcct 480
atgatcaacg aagagattgt ttccgcttta gatgacgtat tgggaaactc tactgaatgg 540
aaaaccactt ctcttgctga tgatttgaaa acaatcattg cgcggacctc taacagagta 600
tttatgggca aagacttaag ccaaaacgaa gactacatca acactgccaa agggttggca 660
atggttgtta tgccagaaac cgtgctccaa gatctcgttc cacaaattct gaaaggacca 720
ctttcaaaga taactagagt gttcaacaac atctatgcga tgaaaagaat cacgtcacat 780
ttgcttcctg tagtaaggca acggtatatt gatgttaaga acgtattcga tggttctgga 840
gacaaagaac agcttcctga caacttgctg acatggatgg tgcaaaagtc aattcgtcga 900
ggagagtcta cagcaaatat cgataaagta ttggtagcgc gtatcggaat ggttaacctg 960
gcagctatcg aaactaccac tgctgccatg actaaaagca tcctagactt ggtcactgtg 1020
ggtgttgaag gaggcttcct ggaagctgtt caggaagaag cattggccgt gttggaagga 1080
tgcaactatc aaccagaaaa gagtgatgtt tcgaaattga atttcaccga aaatgcaatc 1140
aaggagtcac tccgccttca agtcgccttt ccaggtctca tgcgccaagt tgtcagccca 1200
aaaggtgtta cgctagacaa tggtctacac gtgccttacg gtacccgtgt tggtgtatct 1260
gcagctggaa tccatgttga cgactcggtc tatgaaaacg ccacgaccta caatcctggc 1320
agattcatgg tcatagattt agacgctcga ggcaaaccta atccgttatg gaaaggtact 1380
gagaactacc tagcctttgg ccttgggaga cgatcgtgtc ccggccggtg gtatgtgacc 1440
gatcagttaa aactcacact tgcccatatc ttctcgaagt acgagattaa atttgagaag 1500
gctgcaaaga agacaagtgc gctaagaaaa atcctacctg gtgctcctca agatcacgtt 1560
atgattcgac gtagatcgaa agtagccctg tga 1593
<210> 4
<211> 530
<212> PRT
<213> Artificial Sequence
<220>
<223> McfH
<400> 4
Met Val Pro Ser Met Ile Ser Ala Val Val Pro Ile Ile Thr Ser Thr
1 5 10 15
Asn Leu Val Phe Tyr Gly Ala Thr Gly Leu Val Leu Phe Ala Ile Leu
20 25 30
Ala Tyr Ser Leu Asn Arg Leu Thr Thr Trp Glu Tyr Ser Ile Pro Asn
35 40 45
Glu Val Gln Trp Ile Asp Arg Arg Lys Glu Ser Phe Ser Tyr Leu Arg
50 55 60
Ala Lys Ala Arg Ser Leu Val Arg Asn Lys Glu Asn Val Leu Glu Ala
65 70 75 80
Tyr Phe Lys Phe Asn Lys Leu Gly Asn Ala Ala Ala Cys Ala Val Ala
85 90 95
Phe Gly Arg Pro Leu Leu Leu Leu Pro Pro Thr Phe Ile Arg Trp Ile
100 105 110
Val Asp Gln Pro Glu Ser Thr Ile Ser Leu Asp Pro Ile His Asp Asp
115 120 125
Phe His Ala Phe Val Gly Asp Gly Leu Ile Gly Asp His Thr Val Gln
130 135 140
Glu Leu Leu Arg Arg Glu Leu Thr Leu Asn Leu Asp Lys Leu Thr Pro
145 150 155 160
Met Ile Asn Glu Glu Ile Val Ser Ala Leu Asp Asp Val Leu Gly Asn
165 170 175
Ser Thr Glu Trp Lys Thr Thr Ser Leu Ala Asp Asp Leu Lys Thr Ile
180 185 190
Ile Ala Arg Thr Ser Asn Arg Val Phe Met Gly Lys Asp Leu Ser Gln
195 200 205
Asn Glu Asp Tyr Ile Asn Thr Ala Lys Gly Leu Ala Met Val Val Met
210 215 220
Pro Glu Thr Val Leu Gln Asp Leu Val Pro Gln Ile Leu Lys Gly Pro
225 230 235 240
Leu Ser Lys Ile Thr Arg Val Phe Asn Asn Ile Tyr Ala Met Lys Arg
245 250 255
Ile Thr Ser His Leu Leu Pro Val Val Arg Gln Arg Tyr Ile Asp Val
260 265 270
Lys Asn Val Phe Asp Gly Ser Gly Asp Lys Glu Gln Leu Pro Asp Asn
275 280 285
Leu Leu Thr Trp Met Val Gln Lys Ser Ile Arg Arg Gly Glu Ser Thr
290 295 300
Ala Asn Ile Asp Lys Val Leu Val Ala Arg Ile Gly Met Val Asn Leu
305 310 315 320
Ala Ala Ile Glu Thr Thr Thr Ala Ala Met Thr Lys Ser Ile Leu Asp
325 330 335
Leu Val Thr Val Gly Val Glu Gly Gly Phe Leu Glu Ala Val Gln Glu
340 345 350
Glu Ala Leu Ala Val Leu Glu Gly Cys Asn Tyr Gln Pro Glu Lys Ser
355 360 365
Asp Val Ser Lys Leu Asn Phe Thr Glu Asn Ala Ile Lys Glu Ser Leu
370 375 380
Arg Leu Gln Val Ala Phe Pro Gly Leu Met Arg Gln Val Val Ser Pro
385 390 395 400
Lys Gly Val Thr Leu Asp Asn Gly Leu His Val Pro Tyr Gly Thr Arg
405 410 415
Val Gly Val Ser Ala Ala Gly Ile His Val Asp Asp Ser Val Tyr Glu
420 425 430
Asn Ala Thr Thr Tyr Asn Pro Gly Arg Phe Met Val Ile Asp Leu Asp
435 440 445
Ala Arg Gly Lys Pro Asn Pro Leu Trp Lys Gly Thr Glu Asn Tyr Leu
450 455 460
Ala Phe Gly Leu Gly Arg Arg Ser Cys Pro Gly Arg Trp Tyr Val Thr
465 470 475 480
Asp Gln Leu Lys Leu Thr Leu Ala His Ile Phe Ser Lys Tyr Glu Ile
485 490 495
Lys Phe Glu Lys Ala Ala Lys Lys Thr Ser Ala Leu Arg Lys Ile Leu
500 505 510
Pro Gly Ala Pro Gln Asp His Val Met Ile Arg Arg Arg Ser Lys Val
515 520 525
Ala Leu
530

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 a first cytochrome P450 monooxygenase and/or a second cytochrome P450 monooxygenase, and the original strain of the genetically engineered strain is a phoma sphaeroides fungus;
the first cytochrome P450 monooxygenase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity compared to SEQ ID No. 2;
the second cytochrome P450 monooxygenase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity compared to SEQ ID No. 4.
2. A method of preparing/producing micafungin precursor FR901379, comprising the step of culturing the genetically engineered strain of claim 1; preferably, the method further comprises the step of isolating/purifying FR 901379.
3. Use of the genetically engineered strain of claim 1 for the preparation/production of micafungin precursor FR 901379.
4. A method of preparing the genetically engineered strain of claim 1, the method comprising the step of overexpressing the first cytochrome P450 monooxygenase and/or the second cytochrome P450 monooxygenase in a starting strain.
5. The use of the first cytochrome P450 monooxygenase and/or the second cytochrome P450 monooxygenase as claimed in claim 1, or of the genes encoding them, for the preparation of a genetically engineered strain of the high-yielding micafungin precursor FR901379, whose starting strain is a fungus of the genus phoma.
6. Use of a first cytochrome P450 monooxygenase and/or a second cytochrome P450 monooxygenase as claimed in claim 1 or a biological material comprising the first cytochrome P450 monooxygenase and/or the second cytochrome P450 monooxygenase as claimed in claim 1 for the preparation of micafungin precursor FR 901379;
the biological material is selected from the group consisting of: a vector comprising the first cytochrome P450 monooxygenase and/or the second cytochrome P450 monooxygenase of claim 1, or a gene encoding the same; and a host cell comprising the first cytochrome P450 monooxygenase and/or the second cytochrome P450 monooxygenase described in claim 1, or a gene encoding the same.
7. A genetically engineered strain of high-yield WF11899B, wherein the original strain of the genetically engineered strain is a fungus of the genus Sphingomonas, and the genetically engineered strain of high-yield WF11899B is a genetically engineered strain obtained by mutating cytochrome P450 monooxygenase in the original strain; 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 WF11899B has the structure shown in formula II:
8. use of the genetically engineered strain of claim 7 for the preparation/production of WF 11899B.
9. A genetically engineered strain for high yield of a compound shown in a formula IV, wherein an original strain of the genetically engineered strain is a phoma fungus, and the genetically engineered strain is obtained by mutating cytochrome P450 monooxygenase in the original strain; the cytochrome P450 monooxygenase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 4; the compound shown in the formula IV is as follows:
10. use of the genetically engineered strain of claim 9 for the preparation/production of a compound of formula IV.
CN202210570355.9A 2022-05-24 2022-05-24 Strain for high-yield FR901379 and construction method and application thereof Pending CN117143749A (en)

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