Androstenedione substrate-tolerant mutant strain and mutation breeding method thereof

A technology of androstenedione and mutant strains is applied in the field of bioengineering, which can solve the problems of low solubility, low transformation ability of strains, strong hydrophobicity of steroids, etc., and achieves the effect of strong transformation potential and improving the ability of degrading phytosterols

Inactive Publication Date: 2015-04-22
JIANGXI NORMAL UNIV
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  • Abstract
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Problems solved by technology

However, there are still many limiting factors in the transformation process of microorganisms to phytosterols: steroids are highly hydrophobic and have low solu...

Method used

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  • Androstenedione substrate-tolerant mutant strain and mutation breeding method thereof
  • Androstenedione substrate-tolerant mutant strain and mutation breeding method thereof
  • Androstenedione substrate-tolerant mutant strain and mutation breeding method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Diethyl Sulfate and Ultraviolet Combined Mutagenesis of Mycobacterium neogolden:

[0045] Prepare 0.1moL / L K 2 HPO 4 solution (A solution) and 0.1moL / L of KH 2 P0 4 Solution (solution B), take 61.5mL of solution A and 38.5mL of solution B and mix to obtain 100mL of phosphate buffer solution with pH 7.0.

[0046] Use 10mL 0.1moL / L pH7.0 phosphate buffer to wash the slant lawn of Mycobacterium neoaureus JXNU02 cultured for 6 days, pour it into a triangular flask filled with sterile glass beads, shake it fully for 10min, and close it with a stopper. Filter through a sterile funnel with absorbent cotton to obtain a bacterial suspension, which is serially diluted 10 times to reach a concentration of 100-1000cfu / mL for later use.

[0047] Draw 2mL into 5 Erlenmeyer flasks respectively, and add 18mL lmoL / L pH7.0 phosphate buffer. Take the above four Erlenmeyer flasks, add 0.2 mL of diethyl sulfate to each, and finally make a 3% mutagenesis solution.

[0048] ...

Embodiment 2

[0077] Wash the slant lawn of Mycobacterium neoaureus JXNU02 cultivated for 6 days with phosphate buffer solution of pH 7.0, pour it into a triangular flask filled with sterile glass beads, shake it fully for 10 minutes, and use a sterile sterile cotton flask plugged with absorbent cotton. Filter through a funnel to obtain a bacterial suspension, which is serially diluted 10 times to a concentration of 500 cfu / mL for use.

[0078] ② Mutagenesis treatment with 1% DES solution After 10 to 40 minutes of the starting strain of Mycobacterium aureus in the logarithmic growth phase, 0.5 mL of 25% sodium thiosulfate terminator was added, and after 5 to 20 minutes, the culture solution was placed in a sterile petri dish for ultraviolet irradiation. Mutagenesis: Use a 15W UV lamp, preheat for 20-30 minutes in advance, place the bacteria solution at a distance of 20-30cm from the UV lamp and irradiate it for 60-120 seconds.

[0079] ③ Each take 0.1mL of the above-mentioned mutageniz...

Embodiment 3

[0086] Wash the slant lawn of Mycobacterium neoaureus JXNU02 cultured for 6 days with a phosphate buffer solution with a pH of 7.0, filter it with a sterile funnel plugged with absorbent cotton, and serially dilute it by 10-15 times to reach a concentration of 300cfu / mL for later use;

[0087] ②Mutation treatment with 1% DES solution After 10 to 40 minutes of the starting strain of Mycobacterium aureus in the logarithmic growth phase, 0.5 mL of 25% sodium thiosulfate terminator was added, and after 5 to 20 minutes, the culture solution was placed in a sterile petri dish for ultraviolet irradiation. Mutagenesis: Use a 15W UV lamp, preheat for 20-30 minutes in advance, place the bacteria solution at a distance of 20-30cm from the UV lamp and irradiate it for 60-120 seconds.

[0088] Take 0.1mL of the above-mentioned mutagenized bacterial suspension to coat the basic medium and the gradient medium plate, wrap it with black cloth, put it in a 31°C incubator and cultivate it fo...

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Abstract

The invention discloses an androstenedione substrate-tolerant mutant strain and a mutation breeding method thereof. A mutant strain of mycobacterium neoaurum WS3 is preserved in China center for type culture collection with an accession number of CCTCC NO: M 2014322, and the preservation date is July 4, 2014. According to the invention, mycobacterium neoaurumJXNU02 is used as a starting strain; compound mutation is carried out by diethyl sulfate and an ultraviolet mutation method; primary screening is performed by a gradient plate method; secondary screening and verification are carried out by a shake flask fermentation method; and thus the mutant strain of mycobacterium neoaurum WS3 with high substrate tolerance and genetic stability is bred. The method of the invention effectively improves the capability of mycobacteria for degrading phytosterol to generate androstenedione, provides an excellent strain for producing androstenedione by a microbiological method, and has important significance on the pharmaceutical industry of our country.

Description

technical field [0001] The invention relates to the compound mutagenesis of Mycobacterium aureus by using diethyl sulfate and ultraviolet rays, combined with the gradient plate method primary screening technology and shake flask fermentation re-screening technology, to select a substrate that is resistant to substrates and can effectively degrade phytosterol side chains to obtain The new Mycobacterium aureus WS3 of androstenedione ( Mycobacterium neoaurum WS3), which belongs to the technical field of bioengineering. technical background [0002] As an important raw material and key intermediate for the synthesis of steroid hormone drugs, androstenedione (AD) is an important raw material for hormone drugs that the country urgently needs to solve. key". Almost all steroid hormone drugs are produced with androstenedione as the starting material, such as: sex hormones, anabolic hormones, progestins, corticosteroids, dexamethasone, cortisone, etc., which have important commerci...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/01C12N13/00C12R1/32
CPCC12N1/20C12N13/00C12N15/01C12N1/205C12R2001/32
Inventor 王筱兰赵喜华孙婉菊杨慧林张志斌
Owner JIANGXI NORMAL UNIV
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