Squalene engineering strain, squalene synthetic plasmid, cell membrane space expansion plasmid and preparation methods

An engineering strain, squalene technology, applied in botany equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve the problems of limited yield of plant extraction, short growth cycle, low safety of production process, etc. Achieve huge economic benefits, increase synthetic output, and reduce over-exploitation

Active Publication Date: 2020-08-11
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the existing squalene production methods mainly include plant extraction, chemical synthesis and biosynthesis, etc. Plant extraction is limited by the plant cycle and the yield is low; chemical synthesis requires harsh conditions and the production process is low in safety. The cost of industrial synthesis is high; while the method of biosynthesis uses cheap culture medium and microorganisms that are easy to edit and have a short growth cycle. Compared with the harsh reaction conditions and high cost of chemical synthesis, it is cheaper and more efficient.
[0005] At present, foreign scholars have done some pioneering work on the biosynthesis of squalene, but because these methods are not ideal in terms of the yield of strains and target objects, and there are certain limitations, large-scale production cannot be carried out.

Method used

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  • Squalene engineering strain, squalene synthetic plasmid, cell membrane space expansion plasmid and preparation methods
  • Squalene engineering strain, squalene synthetic plasmid, cell membrane space expansion plasmid and preparation methods
  • Squalene engineering strain, squalene synthetic plasmid, cell membrane space expansion plasmid and preparation methods

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Application of embodiment one squalene synthetic plasmid pT-IE in Escherichia coli

[0133] Adopt above-mentioned method to prepare squalene engineering bacterial strain, place squalene engineering bacterial strain SQ-0, SQ-00, SQ-01, SQ-02, SQ-03, SQ-03, SQ-05, SQ-06 respectively in Cultivate in shake flask for 48h, extract squalene. TCL analysis showed a chromatographic band corresponding to the position of the squalene standard.

[0134] See figure 1 , gas chromatographic analysis shows that there is an obvious big peak identical with standard substance at retention time 17.82min, and, gas chromatography-mass spectrometry analysis (GC-MS) is determined to be squalene.

[0135] The SQ-0 and SQ-00 strains were cultivated, and the bacterial cells were collected at 24h, 48h and 72h to measure their cell content and squalene synthesis yield. See figure 2 , there was no significant difference in growth between the two strains, but the squalene production of the SQ-00 ...

Embodiment 2

[0137] The impact of embodiment two cell membrane area size on squalene production in escherichia coli

[0138] The inventor found during the experiment that squalene is fat-soluble and finally stored on the cell membrane, but the limited size of the cell membrane limits the amount of squalene stored. Therefore, the expansion of the membrane area can increase the storage capacity of squalene in the large intestine. Bacteria storage capacity, thereby increasing the production of squalene.

[0139] The SQ-01, SQ-02, SQ-03, SQ-04, SQ-05, SQ-06 strains were cultivated, and the bacterial cells were collected at 24h and 48h respectively to measure their cell content and squalene synthesis yield. See image 3 , the engineered strains SQ-03 and SQ-04 showed significant cell inhibition and reduced squalene production with SQ00. The strains SQ-01, SQ-02, SQ-05, and SQ-06 showed no difference in growth, and the yield was lower than that of SQ-00 at 24h, but all increased at 48h, corres...

Embodiment 3

[0141] The cell membrane area of ​​embodiment squalene engineering bacterial strain SQ-02

[0142] In this example, formaldehyde was used to fix SQ-02 cells, and a transmission electron microscope sample was prepared to observe the morphology and structure of the cell membrane. See Figure 4 , which showed that overexpression of tsr induced expansion of cell membrane structure to increase squalene production more than 2-fold.

[0143] The cell membrane space expansion plasmid disclosed in the present invention can not only be used to increase the cell membrane area of ​​Escherichia coli, thereby increasing squalene production, it can also be applied to other similar fat-soluble products in other embodiments, such as lycopene, shrimp Biological substances such as chlorophyll.

[0144] To sum up: the squalene engineering strain, squalene synthesis plasmid and preparation method thereof of the present invention are a green synthesis method for synthesizing squalene in Escherich...

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Abstract

The invention relates to a squalene engineering strain, a squalene synthetic plasmid, a cell membrane space expansion plasmid and preparation methods. By constructing an engineering strain of a squalene synthetic pathway, developing a microbial cell factory and constructing a squalene synthetic unit of a gene related to membrane area expansion, the biosynthesis and industrial production process ofsqualene is promoted. According to the squalene engineering strain, the squalene synthetic plasmid and the preparation methods thereof, a green synthetic method for synthesizing the squalene in escherichia coli can reduce dependence on natural resources and overdevelopment. Moreover, the method is relatively low in cost, can realize large-scale squalene production, and has huge economic benefits.

Description

technical field [0001] The invention relates to a squalene engineering strain, a squalene synthesis plasmid, a cell membrane space expansion plasmid and a preparation method, belonging to the field of biological genetic engineering. Background technique [0002] Squalene is an unsaturated triterpenoid compound formed by linking 6 isoprenes, and it is a key intermediate product in the human cholesterol metabolism pathway. Squalene widely exists in animals, plants or microorganisms. In animals, the squalene content in the liver of deep-sea sharks is the most abundant. In addition, the content of squalene in yak meat is also relatively rich. The distribution of squalene in plants is also very wide, such as olive oil, pumpkin seeds, etc. are rich in squalene. Squalene has important physiological activities such as strong oxygen-carrying capacity, anti-oxidation, anti-radiation, detoxification, inhibition of microbial growth, regulation of cholesterol metabolism in animals, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/54C12N15/31C12N15/12C12R1/19
CPCC12N9/1085C12N15/70C07K14/395C07K14/245C07K14/47Y02A50/30
Inventor 王崇龙金善元
Owner SUZHOU UNIV
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