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Daptomycin high-producing strain and preparation method thereof

A technology of daptomycin and streptomyces roseospora, which is applied in the field of high-yield daptomycin strains and its preparation, can solve the problems of complex synthesis process, high production cost, and low product yield, and achieve good application prospects, culture The effect of convenient and efficient production

Active Publication Date: 2013-03-13
SICHUAN UNIV
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Problems solved by technology

Domestically, the Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences and Shanghai Gill Biochemical Co., Ltd. successfully synthesized daptomycin (patent application number 200710037006.6), but the synthesis process is complicated, the product yield is low, and the production cost is high, so it is not suitable for daptomycin. industrial production of

Method used

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  • Daptomycin high-producing strain and preparation method thereof
  • Daptomycin high-producing strain and preparation method thereof
  • Daptomycin high-producing strain and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Compound mutagenesis of Streptomyces roseosporus

[0027] 1. EMS mutagenesis

[0028] With Streptomyces roseosporus (Streptomyces roseosporus) NRRL 11379 as the starting strain, NRRL American Agricultural Research Service Culture Collection (Agricultural Research Service Culture Collection). Spores cultured on a slope for 14 days, washed with sterile physiological saline and added to an Erlenmeyer flask containing glass beads, treated at 30°C, shaking at 250 rpm for 2 hours, filtered with sterile gauze to obtain a spore suspension (1×10 6 / Ml). Add 1ml of spore suspension, 9ml of phosphate buffer and 0.1ml of ethyl methanesulfonate (EMS) solution into a 50ml Erlenmeyer flask to make the final concentration of EMS reach 1%, shake at a constant temperature of 30℃, and treat for a certain time (20-60min) ). Then take 0.5ml of the treatment solution, add 0.5ml of 5% sodium thiosulfate solution to wash twice to stop the reaction, and dilute to 2.5×10 3 After spores / ml...

Embodiment 2

[0052] Example 2 Separation and purification of DAP

[0053] Collect the strain Streptomyces roseosporus SCU, fermented 270ml of bacteria liquid for 6-7d in the above example, at 4000rpm, centrifuged for 20min, and collect 250ml of supernatant. Extract twice with n-butanol, 100 ml each time, adjust the pH of the solution to 3.5-4.5, preferably 3.5 with 1 mol / L HCl, and rinse the n-butanol with 30 ml of double-distilled water to maintain the pH at 3.5-4.5, preferably 3.5. Add 100ml of double distilled water to adjust the pH to 6.5-7.3, preferably 7.3, add 1.0g of CaCl2 to the water phase and extract twice with n-butanol, each with 80ml. Collect the n-butanol extract, mix it with an equal volume of double distilled water, adjust the pH 3.5 with 1mol / L HCl, rinse once with 15 ml double distilled water, keep the pH 3.5 to remove Ca 2+ . Remove the water phase, mix the alcohol phase with 50ml of double distilled water, adjust the pH to 7.0 with 1mol / L NaOH, and A-21978C exists in the...

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Abstract

The invention belongs to the field of microbial pharmacy, and in particular relates to a daptomycin high-producing strain and a preparation method thereof. The invention aims to solve the technical problem that daptomycin is low-yield and difficult to be obtained. A technical scheme adopted by the invention to solve the problem provides a mutant strain of Streptomyces roseosporus named after Streptomyces roseosporus SCU and having a preservation number of CGMCC No.6356. The mutant strain can efficiently produce daptomycin, and is easy to be cultured; the preparation process of daptomycin is simple and efficient, can obtain high-purity daptomycin, and has good application prospect in the microbial pharmaceutical industry.

Description

Technical field [0001] The invention belongs to the field of microbial pharmacy, and specifically relates to a high-yield daptomycin strain and a preparation method thereof. Background technique [0002] Anti-drug resistant bacteria drugs are currently one of the focus and development directions of drug research and development in the world. Daptomycin (DAP) is the first new type of cyclic lipopeptide antibiotic in recent years. Its chemical structure and mechanism of action are different from all the existing antibiotics. It is after oxazolidinone antibiotics in the past 40 years. The only new structural category of antibiotics applied to the clinic. Daptomycin was originally obtained by Lilly in the late 1980s. In November 1997, the exclusive global development, production and sales rights of DAP were transferred to Cubist Pharmaceuticals to develop cyclic lipopeptide antibiotics. At the end of 2003, the U.S. Food and Drug Administration (FDA) approved daptomycin for injectio...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N13/00C12N15/01C07K7/08C07K1/18C07K1/16C12R1/465
Inventor 梁淑芳李国顺马雯李蓉晖
Owner SICHUAN UNIV
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