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Production method of squalene

A technology for squalene and squalene synthase, applied in the field of genetic engineering, can solve the problems of high price of squalene, high unit cost, etc., and achieves a technology that is conducive to large-scale application, simple production method, and increased output. Effect

Active Publication Date: 2014-09-17
QINGDAO VLAND BIOTECH GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the problem of high unit cost has not been solved, so that the price of squalene remains high, resulting in squalene and squalane (perhydrogenated derivatives of squalene) are currently only small-scale market applications, such as watch lubricants, Chemical intermediates for pharmaceuticals / nutraceuticals, cosmetics, perfumes and high-value products

Method used

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  • Production method of squalene
  • Production method of squalene
  • Production method of squalene

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Experimental program
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Effect test

Embodiment 1

[0020] The construction of embodiment 1 squalene synthase expression vector

[0021] In order to enable the squalene synthase gene to be transcribed more efficiently in Escherichia coli, the codons of the squalene synthase gene GenBank Accession No.GM732820 in the Indian ginseng genome were first optimized, and obtained after long-term research The nucleotide sequence is the squalene synthase gene of SEQ ID NO: 2, and the encoded amino acid sequence is SEQ ID NO: 1. The gene nucleotide fragment whose sequence is SEQ ID NO: 2 was synthesized by Shanghai Yingwei Jieji Biotechnology Co., Ltd. as a template for amplification.

[0022] Design restriction sites BamHI and EcoRI on both sides of the gene fragment. Using primer F respectively:

[0023] 5′-CGC GGATCC ATGGGCACCCTGCGTGCAAT-3' and primer R:

[0024] 5′-CCG GAATTC TTAATTCGGCTCGCTGCGAATCA-3′ was amplified by PCR, PCR amplification conditions: 95°C, 10min; 95°C, 30S; 55°C, 30S, 30 cycles; 72°C, 1.5min; 72°C, 10min.

...

Embodiment 2

[0026] Embodiment 2 Construction of squalene synthase recombinant expression engineering bacteria

[0027] Positive transformants were cultured in LB medium at 37°C and 200rpm for 8h, the cells were collected, plasmid SQS-pET28a(+) was extracted, and stored at 4°C until use.

[0028] Prepare Escherichia coli E.coli BL21 transformation competent cells, add SQS-pET28a(+) plasmid, mix well, place in ice bath for 30min, heat shock at 42°C for 90s for transformation; add 1ml LB medium, rejuvenate at 37°C for 1h, collect bacteria The body was smeared on a resistance screening LB plate with a kanamycin concentration of 50 μg / ml, and cultured overnight at 37°C. The target gene SQS was verified by colony PCR, and recombinant strains of Escherichia coli were constructed.

[0029] Ferment and culture the recombinant strain in LB fermentation medium at 37°C and 220rpm overnight; transfer to fresh LB medium every other day, culture at 37°C for 2h; add final concentration of 1mM IPTG, indu...

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Abstract

The invention relates to a production method of squalene, and in particular relates to a method for cloning a squalene synthetase gene and co-expressing in escherichia coli so as to establish a recombinant strain capable of synthesizing squalene. The production method of squalene, provided by the invention, is simple and efficient, the established escherichia coli engineering bacteria can be used for producing squalene with a high yield and can be widely applied to the fermentation production of squalene, the output of squalene is increased, and then the large-scale application of squalene is facilitated.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a squalene production method, namely a squalene biosynthesis method, and a constructed recombinant bacterial strain. technical background [0002] Squalene is a lipid unsaponifiable substance, originally discovered from shark liver oil, named Sqμalene in 1914, and its chemical name is 2,6,10,15,19,23-hexamethyl-2 ,6,10,14,18,22-Tetracosahexaene, an open-chain triterpene, also known as cod liver oil terpene, can improve the activity of superoxide dismutase (SOD) in the body, enhance the body's immunity, and improve sexual function , anti-aging, anti-fatigue, anti-tumor and other physiological functions, it is a non-toxic marine bioactive substance with the effect of preventing and curing diseases. [0003] As the main source of squalene, deep-sea shark cod liver oil needs to be obtained by hunting a large number of deep-sea sharks, which leads to a series ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P5/02C12N15/70C12N1/21C12R1/19
Inventor 王华明郭小飞李玮涛
Owner QINGDAO VLAND BIOTECH GRP
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