Method for screening high-yield amphotericin B streptomyces nodosus through high-throughput mutagenesis and strain

A technology for amphotericin and mutagenesis screening, applied in microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve the problem of V-shaped pore not penetrating the cell membrane, etc., to improve the efficiency of screening or verification and the overall yield The effect of raising and lowering costs

Active Publication Date: 2019-12-13
ZHEJIANG UNIV OF TECH
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  • Description
  • Claims
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Problems solved by technology

[0005] Studies have shown that amphotericin B initially aggregates on the fungal cell membrane and inserts into the cell membrane to form a V-shaped pore. The amphotericin B molecule tilts so that the terminal OH group on the polyene lactone ring faces the center of the lipid bilayer, but this process does not No sterol is involved, and the V-shaped channel does not penetrate the cell membrane, such as figure 1

Method used

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  • Method for screening high-yield amphotericin B streptomyces nodosus through high-throughput mutagenesis and strain
  • Method for screening high-yield amphotericin B streptomyces nodosus through high-throughput mutagenesis and strain
  • Method for screening high-yield amphotericin B streptomyces nodosus through high-throughput mutagenesis and strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Construction of high-throughput screening method

[0053] (1) Spread or streak the mutagenized spores and the control strain Streptomyces nodosum N3 (ATCC14899) on the GYM solid medium, and culture at 26° C. for 7 days until a single colony can be observed. Using a sterile toothpick or pipette tip, prick a single colony and streak a new GYM solid medium plate. After numbering, culture at 26°C for 4-7 days for preservation. The plates after the retention treatment were used for the inhibition zone experiment.

[0054] (2) Using yeast X33 (preserved in the laboratory) as a sensitive strain, pick a single colony on the GYM solid medium, culture it in the GYM liquid medium for 24 hours, take 100 μL and spread it on the GYM solid medium. On the plate that has been preserved in (1), use a sterile puncher or a pipette tip to pick out agar blocks containing a single colony, place them upside down on the GYM solid medium that has been coated with yeast, and number t...

Embodiment 2

[0055] Embodiment 2: the mutagenic strain of high AmB production

[0056] (1) Streptomyces nodosus N3 (ATCC14899) was inoculated on a GYM plate and cultured at 26°C for 7 days, and the gray-black spores were taken, and the surface spores were eluted into 10 mL of sterile water with a cotton swab, and washed The spore suspension was filtered with a syringe containing cotton, and the filtered spores were centrifuged at 12000rpm for 5min to remove the supernatant, added 10mL of sterile water to resuspend, centrifuged at 12000rpm for 5min to elute again, and resuspended with 5mL of sterile water as spore suspension.

[0057] (2) NTG-UV mutagenesis method: take 10mL of diluted spore suspension and place it under a UV lamp (15W, 254nm) at 20cm for 1min, inoculate it in GYM medium, and incubate at 26°C for 24-32h in the dark , collect the cells after mutagenesis, and treat them with NTG. The treatment method: use 50mM PBS buffer solution containing 5mg / mL NTG per 1mL of cell suspens...

Embodiment 3

[0064] Embodiment 3: Shake flask fermentation produces AmB

[0065] (1) Preparation of seed liquid: streak the Streptomyces nodosum mutagen (i.e. CCTCC NO: M 2017426) with high amphotericin B production prepared in Example 2 onto a GYM plate, culture at 26°C for 4 days, and select a single colony , inoculated into the seed medium, cultured at 26° C., 220 rpm for 48 hours, to obtain seed liquid.

[0066] The seed medium was prepared as follows: peptone 20g, NaCl 8g, glucose 15g, yeast powder 10g, CaCO 3 1g, add water to make up to 1L, pH 7.0, sterilize at 121°C for 20min.

[0067] (2) Fermentation culture

[0068] Load 100mL of fermentation medium into a 500mL shake flask, inoculate the seed solution at a volume concentration of 4% during fermentation, and ferment and cultivate at 26°C and 220rpm for 144 hours. The AmB production in the fermentation broth can reach 12g / L.

[0069] Fermentation medium composition: glucose 70g / L, beef extract 8g / L, soybean protein powder 8g / L,...

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Abstract

The invention relates to a method for screening high-yield amphotericin B streptomyces nodosus through high-throughput mutagenesis, a screened mutant strain, a recombinant engineering bacterium obtained from the mutant strain and an application thereof. The streptomyces nodosum ZJB2016050 which is a streptomyces nodosum strain capable of highly yielding amphotericin B is obtained by screening in the invention. The method disclosed by the invention has the beneficial effects that 1, the yield of amphotericin B can be conveniently, quickly and efficiently detected by a high-throughput amphotericin B high-yield strain screening method, and the mutation breeding and screening or verification efficiency of genetic engineering strains can be improved; 2, compared with an original strain N3, thestreptomyces nodosus N5 obtained by mutation breeding has the advantages that the amphotericin B yield is increased by 20%, and the streptomyces nodosus N5 can be used as the original strain of genetically engineered bacteria; 3, the yield of amphotericin B is respectively increased by overexpression of functional genes, wherein the yield of amphotericin B is increased by 20% by acetyl coenzyme Acarboxylase 1 (acc1), and 4, the kanamycin is low in price, wide in antibacterial spectrum and strong in bactericidal effect, is suitable for industrial use, has a relatively strong effect of improving the total yield of enterprises, and can reduce the cost by about 2000 yuan per tank by taking a 5L tank in a laboratory as a calculation result.

Description

[0001] (1) Technical field [0002] The invention relates to a high-throughput mutagenesis screening method for Streptomyces nodosa high-yielding amphotericin B, the screened mutagenic strain, the recombinant engineering bacteria obtained from the mutagenic strain and its application. [0003] (2) Background technology [0004] Polyene macrolide antibiotics are a class of antibiotics mainly composed of Streptomyces secondary metabolites. This type of antibiotic has broad-spectrum antifungal activity and little tolerance, and has become the most effective antifungal drug so far, and is widely used in the treatment of various infectious diseases caused by fungi. Amphotericin B (Amphotericin B, AmB) was first discovered in 1955. Amphotericin B was first isolated from Streptomyces nodosus in water and soil samples from the Orinoco River in Venezuela. Amphotericin B was extracted from the fermentation broth of Streptomyces tuberculosis in 1959, and it began to be marketed in 1966. ...

Claims

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Application Information

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IPC IPC(8): C12N13/00C12N15/01C12N1/21C12P19/62C12R1/465
CPCC12N9/12C12N9/90C12N9/93C12N13/00C12N15/01C12P19/62C12Y501/99001C12Y504/99002C12Y604/01002
Inventor 柳志强郑裕国黄恺张博姜圣贤张雨函陈燏
Owner ZHEJIANG UNIV OF TECH
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