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Genetic recombination strain for producing pneumocandins B0, breeding method and application

A gene recombination and gene rearrangement technology, applied in the field of microorganisms, can solve problems such as separation and extraction difficulties

Active Publication Date: 2015-04-22
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main structural analog A 0 and C 0 It has caused great difficulties to the later separation and extraction
Merck conducted mutagenesis selection on the original strain ATCC20868, and obtained Newmocontin B 0 High-yield strain ATCC20957, but the pneumocantine B of this strain 0 The yield is only 285mg / L, and pneumocidine A 0 The content is higher, the ratio of the two concentrations (B 0 / A 0 ) is 10

Method used

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  • Genetic recombination strain for producing pneumocandins B0, breeding method and application
  • Genetic recombination strain for producing pneumocandins B0, breeding method and application

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Experimental program
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Effect test

preparation example Construction

[0038] The preparation method of the enzymolysis solution is as follows: take 80g of Yatalse, 80g of wall-melting enzyme, and 80g of helicase, and dissolve them with 1L of hypertonic buffer I.

[0039] Pretreatment of fermentation broth:

[0040] Take 1ml of fermentation broth, add 9ml of ethanol, shake vigorously for 10min, then centrifuge at 3000rpm for 10min, and the supernatant after centrifugation is used for further detection.

[0041] Neomercontin A 0 , Neomercantin B 0 detection of:

[0042] Adopt Diane high performance liquid chromatography U3000 to measure, and its specific method is: chromatographic column, C18 post; Mobile phase A, acetonitrile; Mobile phase B, the phosphoric acid of 0.1%; Elution program, 0min-20min, A:B=40: 60, 20min-40min, A:B=50:50; flow rate 1.5ml / min, detection wavelength, 210nm; detection temperature, 25°C.

[0043] Neomercontin C 0 detection of:

[0044] Determination by Dionne high performance liquid chromatography P680. The specifi...

Embodiment 1

[0045] The breeding method of embodiment 1 gene recombinant strain Glarea lozoyensis

[0046] Starting strain: Glarea lozoyensis 74030 purchased from American Type Culture Collection (ATCC).

[0047] (1) Preparation of protoplasts

[0048] The most vigorous growth after purification, pneumocantine B 0 Put the strain of Glarea lozoyensis with the highest yield into a 250ml shaker flask containing 50ml of PDA liquid medium, and culture it at 26°C and 220r / min for 3 days, take 8ml of the culture solution into a sterile 10ml centrifuge tube, and set it at 1800r / min , 4°C, centrifuge for 10min to remove the supernatant, wash twice with hypertonic buffer I, collect the mycelium, add 2ml of newly prepared enzymatic hydrolysis solution to the collected mycelium, and cultivate at 26°C with shaking at 100r / min 3h, get the enzymolysis liquid; take a 2ml centrifuge tube, add 0.2ml of 1.2M sucrose solution to the centrifuge tube, add the enzymolysis liquid into it, centrifuge at 1800rpm,...

Embodiment 2

[0065] Embodiment 2 Utilizes Glarea lozoyensis Q45 to carry out pneumocandine B on 5L fermentation tank 0 method of production.

[0066] 1. Inclined Seed Culture

[0067] Solid culture method: the strains were inoculated on PDA solid slant medium and cultured at 25°C for 11 days.

[0068] 2. Shake Flask Seed Culture

[0069] Seed medium (g / L): glucose 40, soybean powder 20, corn steep liquor 10, KH 2 PO 4 1. FeSO 4 ·7H 2 O 0.01, MnSO 4 4H 2 O0.01, ZnSO 4 ·7H 2 O 0.002, CaCl 2 2H 2O 0.001, CuCl 2 2H 2 O 0.00025, H 3 BO 3 0.00056, (NH 4 ) 6 Mo 7 o 24 4H 2 O 0.0002, pH adjusted to 5.0.

[0070] Culture temperature: 25°C

[0071] Culture time: 3 days

[0072] Shaker speed: 220rpm

[0073] Shake flask seed culture method: remove a 1cm piece from the solid slant medium 2 Bacteria with large and small sizes were inserted into 250ml shake flasks with a filling volume of 50ml, and cultivated for 3 days.

[0074] 3. Fermentation in 5L fermenter

[0075] 5L fer...

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Abstract

The invention discloses a genetic recombination strain for producing pneumocandins B0, a breeding method and applications. The bacterial strain classification name of the genetic recombination strain is Glarea lozoyensis Q45; the preservation registration number is CCTCC NO: M2014416; and the preservation date is September 14, 2014. The method for breeding the strain includes the steps that an original strain is manufactured into a protoplast; a mutation library composed of a plurality of mutant strains is obtained through ion implantation and lithium chloride processing; the mutation library is manufactured into a protoplast again; random fusion is carried out on the protoplasts after ion implantation inactivation and heat inactivation are carried out; fermentation screening is carried out on high-yield genetic recombinant bacteria; and protoplast preparation and fusion and fusant screening are carried out on the screened genetic recombinant bacteria, and therefore the genetic recombination strain is obtained. The genetic recombination strain is excellent in performance and stable in production capacity; the yield of the pneumocandins B0 obtained through fermentation is 6 g / L; in addition, by-products are few; the post-extraction difficulty of the pneumocandins B0 is reduced; the application to high-quality caspofungin preparation is facilitated; and the genetic recombination strain has the important industrial value.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a method for producing pneumocidine B 0 Gene recombinant strains and their breeding methods and applications. Background technique [0002] Echinocandin antifungal drugs are a new type of lipopeptide compounds, which inhibit the synthesis of fungal cell walls by non-competitively inhibiting the unique β-1-3-glucan synthase activity in fungal cell walls, and are not harmful to humans. make an impact. This is obviously different from the mechanism of action of traditional antibiotics such as amphotericin, which makes echinocandins have the characteristics of strong broad-spectrum, low drug resistance, and small drug-drug interactions. The development direction of broad-spectrum new antibiotics. [0003] Caspofungin is the first approved echinocandin antifungal drug, which consists of the secondary metabolite nemocontin B of the filamentous fungus Glarea lozoyensis 0 Derived from c...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N13/00C12N15/04C12P21/04C12R1/645
CPCC12N1/14C12N13/00C12N15/04C12P21/02C12N1/145C12R2001/645
Inventor 黄和宋萍沈文和秦婷婷章人川纪晓俊
Owner NANJING UNIV OF TECH
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