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Engineered strain of streptomyces clavuligerus, and preparation method and application thereof

A technology of Streptomyces coryneformis and strains, applied in the biological field, can solve the problems of increasing the yield of metabolites and the like, and achieve the effects of stable acid production, convenient genetic engineering modification, and increased yield

Inactive Publication Date: 2013-04-03
TIANJIN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Blockade of one metabolite in Streptomyces clavulicularis may lead to increased production of other metabolites, study suggests

Method used

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  • Engineered strain of streptomyces clavuligerus, and preparation method and application thereof
  • Engineered strain of streptomyces clavuligerus, and preparation method and application thereof
  • Engineered strain of streptomyces clavuligerus, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Ultraviolet mutagenesis of Streptomyces clavulicularis NRRL3585

[0057] Strain: Streptomyces clavulatus NRRL3585 ( S. clavuligerus NRRL3585), strain collection number CPCC 600044; Klebsiella pneumoniae ( Klebsiella pneumoniae ) ATCC 29665, strain preservation number 29665?.

[0058] Main medium and solution: ISP medium: ISP 8g, agar powder 20g, adjust pH to 7.3; TSB liquid medium (L -1 ): TSB 30g, starch 10g, pH6.8~7.0; SS liquid medium (L -1 ): soybean flour 15g, starch 4.7g, KH 2 PO 4 0.1g, FeSO 4 ·7H 2 O 0.2g, pH 6.8.

[0059] For other conventional media and solutions, refer to Sambrook J, Fritsch EF, Maniartis T. Molecular Cloning Experiment Guide [M], Third Edition, Beijing: Science Press, 2002.

[0060] method:

[0061] Preparation of spore suspension

[0062] (1) Pick cultured on ISP solid medium S. clavuligerus The single colonies were transferred to the YMGA slant, cultured at 28°C for 2-3 days, scraped off the spores with a large inoculation...

Embodiment 2

[0112] PCR Amplification of lat Gene of Streptomyces clavulicularis

[0113] Strain: Streptomyces clavulatus strain B372 induced by ultraviolet mutagenesis, preserved in our laboratory (the strain constructed in Example 1).

[0114] Main medium and solution: ISP, YMGA medium refer to Example 1.

[0115] For other conventional media and solutions, refer to Sambrook J, Fritsch EF, Maniartis T. Molecular Cloning Experiment Guide [M], Third Edition, Beijing: Science Press, 2002.

[0116] method:

[0117] Extraction of Genomic DNA from Streptomyces clavulicularis:

[0118] (1) Pick the solid YMGA plate S. clavuligerus B372 single colony was inoculated in liquid ISP medium, cultured at 28°C, 200r / min for 2 days, and centrifuged at 2000r / min for 5min to harvest the bacteria.

[0119] (2) Wash once with 10mM EDTA pH8.0, or wash twice with water, and store the obtained cells at -20°C.

[0120] (3) Add SET solution to 1 mL, add 20 μL of 50 mg / mL lysozyme, and bathe in water at 3...

Embodiment 3

[0137] PCR amplification of AMP resistance cassette

[0138] Strain: Streptomyces clavulicularis strain B372, constructed in Example 1, by ultraviolet mutagenesis.

[0139] Plasmid: Plasmid pIJ773 containing AMP resistance gene, purchased from China Plasmid Vector Strain Cell Line Gene Collection Center.

[0140] method:

[0141] According to Streptomyces clavulatus lat The sequence of the gene and the sequence of the Amp resistance gene in the plasmid pIJ773 (the sequence also contains the transfer origin of replication oriT and the recognition site FRT sequence of FLP recombinase, oriT To facilitate the conjugation and transfer of recombinant plasmids later) design a pair of 59nt and 58nt long primers, which contain the complementary sequences of the FRT sequences on both sides of the AMP resistance gene of 20nt and 19nt respectively and lat Two 39nt complementary sequences upstream and downstream of the gene ( Figure 4 );

[0142] The sequences of the two p...

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Abstract

The invention discloses an engineered strain of streptomyces clavuligerus, and a preparation method and application thereof. Through treating streptomyces clavuligerus by using an ultraviolet mutagenesis technology firstly, a mutant strain B372 is obtained, wherein the clavulanic acid yield of the mutant strain B372 is 1.9 times as large as that of an original strain; and then, through taking the mutant strain as a starting strain, a mutant strain S.clavuligeruslat:: Delta subjected to lat gene knockout is built by using PCR-TARGETING. Studies show that after ultraviolet mutagenesis and lat gene knockout, the clavulanic acid yield of the streptomyces clavuligerus is significantly increased and is 2.3 times as large as that of the original strain. The clavulanic acid yield of the engineered strain disclosed by the invention is significantly increased, and the engineered strain does not contain any resistance makers, therefore, the engineered strain is extremely conveniently used for further genetic engineering reconstruction; and through continuous passage, the clavulanic acid yield is relatively stable and good in repeatability.

Description

[0001] This application is supported by Tianjin Normal University Doctoral Fund (52x09010). technical field [0002] The invention belongs to the field of biological technology, and in particular relates to an ultraviolet mutagenesis and gene blocking mutant bacterium of Streptomyces clavulus and a preparation method thereof. Background technique [0003] For decades, due to the extensive use of antibiotics, bacteria have become resistant to many drugs, and the resistance of pathogenic bacteria to β-lactam antibiotics has become one of the important factors for clinically refractory infections. [0004] Clavulanic acid (clavulanic acid) is a Streptomyces clavulatum ( Streptomyces clavuligerus ), also known as clavulanic acid, is the first β-lactamase inhibitor discovered and used clinically. Clavulanic acid itself has weak antibacterial activity, but it has potent, broad-spectrum and irreversible β-lactamase inhibitory activity. Clavulanic acid is combined with β-lactam an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N13/00C12N15/01C12N15/09A61K35/74A61P31/04C12R1/465
Inventor 左志晗
Owner TIANJIN NORMAL UNIVERSITY
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