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A genetic engineering strain for increasing the yield of ascomycin and a constructing method

A technology of genetically engineered strains and ascomycin, which is applied in the field of genetic engineering, can solve the problems of unreported patent research on ascomycin genetically engineered strains, and achieve significant application value, increase production, and reduce costs

Inactive Publication Date: 2015-07-08
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the construction methods of Streptomyces genetically engineered strains are becoming more and more perfect, but there are few studies on the genetically engineered strains of Streptomyces hygroscopicus subsp. Patent research has not been reported

Method used

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  • A genetic engineering strain for increasing the yield of ascomycin and a constructing method
  • A genetic engineering strain for increasing the yield of ascomycin and a constructing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Construction of double-gene tandem overexpression vector pIBNR

[0055] Using the genome extracted from the S.hygroscopicus var.ascomyceticus mycelia as a template, design primers to PCR amplify the fkbN and frr gene fragments, and detect the resulting PCR products by 0.8% agarose gel electrophoresis, and the sizes are 2700bp , 500bp electrophoresis band. After the frr gene amplified fragment was purified, digested and recovered, it was ligated with the corresponding digested pUC18 plasmid, transformed into DH5α Escherichia coli competent cells, positive transformants were screened on the ampicillin resistance plate, the plasmid was extracted, and the restriction endogenous Dicer NdeI and XbaI were used for double-digestion verification. After double-digestion, a fragment of about 500 bp was obtained by agarose gel electrophoresis, indicating that a recombinant plasmid with the correct insertion sequence was obtained, named pUCR, and transformed into the bact...

Embodiment 2

[0067] Example 2: S.hygroscopicus var.ascomyceticus-E.Coli ET12567 (pIBNR) genera indirect conjugative transfer experiment

[0068] (1) The donor bacterium is E.coli ET12567 (PU28002) containing the overexpression vector pIBNR, and the recipient bacterium is fresh spores of Streptomyces hygroscopicus.

[0069] (2) Inoculate 500 μL E. coli donor into 10 mL of fresh LB medium containing corresponding antibiotics (50 μg / mL apramycin, 25 μg / mL kanamycin, 25 μg / mL chloramphenicol), overnight at 37 °C Cultivate; inoculate 1 mL of the overnight culture into 50 mL of fresh LB containing the corresponding antibiotics, and culture at 37°C until OD 600 0.4-0.6, 4000r / min centrifuge 3min to collect the bacteria,

[0070] (3) Wash the bacteria twice with an equal volume of fresh LB (to wash off the antibiotics), and finally suspend with 2 mL of LB liquid medium.

[0071] (4) Scrape off the fresh spores of Streptomyces hygroscopicus, prepare a single spore suspension, wash once with 500 μ...

Embodiment 3

[0080] Example 3: Fermentation experiment of ascomycin-producing engineering strain Streptomyces hygroscopicus var.ascomyceticus TD01

[0081] (1) culture medium

[0082] Seed liquid medium (g / L): starch 10, glucose 30, peptone 6, yeast powder 6, CaCO 3 2, pH 7.0

[0083] Fermentation medium (g / L): 20 starch, 40 dextrin, 5 peptone, 5 yeast powder, 5 corn steep liquor, CaCO 3 1. MgSO 4 ·7H 2 O 1,MnSO 4 ·H 2 O 0.5, (NH 4 ) 2 SO 4 1.5,K 2 HPO 4 ·3H 2 O 0.5, soybean oil 11.

[0084] (2) Fermentation experiment: Scrape the spores of the high-efficiency ascomycin-producing genetically engineered strain Streptomyces hygroscopicus var.ascomyceticus TD01 obtained by the present invention and the spores of the starting strain Streptomyces hygroscopicus from an inoculation loop respectively, and inoculate into 250 mL of shaker of 40 mL seed culture medium. In the bottle, 28 ℃, 220rpm shaking table culture 60h, prepare seed solution; According to the volume ratio is 10% inocu...

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Abstract

The invention relates to a genetic engineering strain for increasing the yield of ascomycin and a constructing method thereof. The genetic engineering strain is streptomyces hygroscopicus var.ascomyceticus TD01, and is deposited in the China General Microbiological Culture Collection Center with an accession number of CGMCC 10615. By cloning fkbN and frr genes, an fkbN-frr double-gene tandem overexpression vector is constructed by utilization of escherichia coli-streptomycete shuttle plasmid pIB139 and is transformed into escherichia coli ET12567, and streptomyces hygroscopicus var.ascomyceticus is transformed by a conjugal transfer method to allow the overexpression vector to be expressed in an original strain. The yield of the ascomycin of the constructed ascomycin genetic engineering strain TD01 reaches 500 mg / L, and is increased by 42% than that of the original strain. The genetic engineering strain and the constructing method have high potential and application value.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to constructing a double-gene tandem overexpression vector and expressing it in Streptomyces hygroscopicus subsp. Application in fermentation production. In particular, it relates to a genetically engineered strain and a construction method for improving the production of ascomycin. Background technique [0002] Ascomycin (FK-520) is a macrolide antibiotic composed of 23-membered rings produced by the fermentation of Streptomyces hygroscopicus var.ascomyceticus. It is a natural compound synthesized by polyketide synthase / non-ribosomal synthetase and has antifungal and immunosuppressive activities. Ascomycin was first isolated from the fermentation product of S. hygroscopicus var ascomyceticus and was considered as an antifungal antibiotic. Ascomycin was found to have immunosuppressive characteristics when studying the analogs of low toxicity FK506. Ascomyci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/76C12N15/31C12R1/55
Inventor 闻建平宋柯璟齐海山
Owner TIANJIN UNIV
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