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Saccharopolyspora spinosa rhamnose biosynthesis gene duplication engineering strain

A technology of Saccharopolyspora spinosa and biosynthesis, applied in the direction of microorganisms, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of low success probability and technical difficulties of protoplast transformation technology and conjugation transfer technology

Inactive Publication Date: 2015-10-07
HUNAN NORMAL UNIVERSITY
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Problems solved by technology

[0005] However, the existing technologies for integrating genes into the S. spinosa genome are relatively difficult, such as protoplast transformation technology and conjugative transfer technology, which have relatively low success rates.

Method used

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  • Saccharopolyspora spinosa rhamnose biosynthesis gene duplication engineering strain
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  • Saccharopolyspora spinosa rhamnose biosynthesis gene duplication engineering strain

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Embodiment 1

[0026] The Saccharopolyspora spinosa S. sp-RM and Saccharopolyspora spinosa S. sp-RM were typically cultured in China on June 8, 2015 in the Saccharopolyspora spinosa S. Culture Collection Center (CCTCC for short, address: Wuhan University, Wuhan, China), the strain collection number is CCTCC NO: M 2015363.

[0027] (1) Specific medium formula and culture conditions

[0028] (1) S. spinosa Seed activation uses SP-2 medium: glucose 9 g / L, Tryptic Soy Broth 30 g / L, yeast extract 3 g / L, magnesium sulfate 2 g / L. Inoculate the monoclonal or 1% strain preservation solution into a 250 mL shake flask filled with 20 mL SP-2, and incubate at 30°C and 300 r / min for 48-72 h;

[0029] (2) TSB medium was used for transfer: Tryptic Soy Broth 30 g / L. Inoculate 1% bacterial solution into a 250 mL shake flask filled with 20 mL SP-2, and incubate at 30°C and 300 r / min for 48-72 h;

[0030] (3) SP-3 medium was used for fermentation culture: glucose 60 g / L, soluble starch 10 g / L, corn steep l...

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Abstract

The invention relates to a Saccharopolyspora spinosa rhamnose biosynthesis gene duplication engineering strain (S. sp-RM, Saccharopolyspora spinosa) which is collected by China Center for Type Culture Collection on June 8th, 2015, and the culture collection number is CCTCC NO:M2015363. The PCR (polymerase chain reaction) identification result indicates that the plasmid is successfully integrated to the Saccharopolyspora pogona genome. The real-time fluorescent quantitative PCR result indicates that the transcription levels of genes gdh, kre, epi and gtt in the engineering strain are respectively enhanced by 80.3 times, 30.8 times, 23.8 times and 18.3 times as compared with the initial strain. The HPLC (high performance liquid chromatography) detection result indicates that the pleocidin yield of the engineering bacterium S. sp-RM is enhanced by 73% as compared with the initial strain.

Description

technical field [0001] The invention relates to a doubling engineering strain of the rhamnose biosynthesis gene of Saccharopolyspora spinosa, which uses genetic engineering technology to increase the copy number of the rhamnose biosynthesis gene in Saccharopolyspora spinosa to obtain a strain S with high germicidin production . sp-RM. Background technique [0002] Spinosyn and its derivatives have both high-efficiency insecticidal activity and excellent green and safe characteristics, and have become a hot spot in the field of biological insecticide research in recent years. However, the extremely low fermentation yield of wild strains hinders the further development of spinosyns. research problem. Spinosad depolarizes insect nerve cells by binding to nicotinic acetylcholine receptors, causing hyperactivation of the central nervous system, resulting in non-functional muscle contraction, failure and paralysis, so it has rapid contact and ingestion toxicity to insects. Spino...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12P19/62C12R1/645
Inventor 夏立秋杨燕丁学知孙运军
Owner HUNAN NORMAL UNIVERSITY
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