Schizochytrium limacinum genetic engineering strain for expressing EPA synthase gene as well as construction method and application of schizochytrium limacinum genetic engineering strain

A genetically engineered strain, the technology of Schizochytrium, applied in the field of Schizochytrium genetically engineered strains, can solve the problems of unstable fish quantity, EPA output and quality, hidden dangers to human health and safety, and inability to satisfy consumers, etc., reaching EPA Increased synthesis yield, short culture period, and low production cost

Pending Publication Date: 2022-05-13
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] (1) Restricted by season and place of production, the quantity of fish and the yield and quality of EPA are unstable;
[0005] (2) Due to the deterioration of the ecological environment, marine pollution is becoming more and more serious, and pollutants enter the human body through fish oil, which poses a safety hazard to human health;
[0006] (3) EPA extracted from fish oil has a strong fishy smell, which is not easily accepted by consumers;
[0007] (4) The purification cost is high and the product price is expensive
[0008] Therefore, the way to obtain EPA from marine fish cannot meet the requirements of consumers in terms of the total market demand or the quality of fish oil products.

Method used

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  • Schizochytrium limacinum genetic engineering strain for expressing EPA synthase gene as well as construction method and application of schizochytrium limacinum genetic engineering strain
  • Schizochytrium limacinum genetic engineering strain for expressing EPA synthase gene as well as construction method and application of schizochytrium limacinum genetic engineering strain
  • Schizochytrium limacinum genetic engineering strain for expressing EPA synthase gene as well as construction method and application of schizochytrium limacinum genetic engineering strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] The construction of embodiment 1 recombinant plasmid pfaAB-pzpk-NeoR-TE

[0056] This embodiment is a method for constructing the recombinant plasmid pfaAB-pzpk-NeoR-TE, including the following steps in sequence:

[0057] S1. Construction of recombinant plasmid pzpk-NeoR

[0058] S11. Using the plasmid pbacNeoR as a template, using NeoR-F (gene sequence as shown in SEQ ID NO.5) and NeoR-R (gene sequence as shown in SEQ ID NO.6) as primers to perform PCR amplification to obtain the NeoR gene (neomycin resistance gene), the nucleotide sequence of the NeoR gene is shown in SEQ ID NO.7;

[0059] The PCR program is as follows: denaturation at 98°C for 10 s, annealing at 56°C for 10 s, extension at 72°C for 1 min (extension time = target fragment length / 1 kb, unit min), repeat 35 cycles;

[0060] S12. Using the Schizochytrium sp.HX-308 genome as a template, and using P2845-F (gene sequence as shown in SEQ ID NO.8) and P2845-R (gene sequence as shown in SEQ ID NO.9) as prime...

Embodiment 2

[0117] Example 2 Construction of recombinant plasmid pfaCD-pCTS-BleoR-TE

[0118] S1. Construction of recombinant plasmid pCTS-BleoR

[0119] S11. Using the plasmid pPICZaA as a template, using BleoR-F (as shown in SEQ ID NO.21) and BleoR-R (as shown in SEQ ID NO.22) as primers to carry out PCR amplification to obtain the BleoR gene expression cassette (including the promoter sub TEF-1, terminator CYC1t) (SEQ ID NO.23);

[0120] S12. Then use EcoRI to double digest the pCTS plasmid. Then, the digested pCTS plasmid backbone and the BleoR gene expression cassette obtained by the above PCR amplification were cloned in one step using the ClonExpress MultiS One Step Cloning Kit, and the constructed plasmid was named pCTS-BleoR;

[0121] S13. The circular recombinant vector was transformed into Escherichia coli DH5a competent cells, and the positive recombinant plasmid pCTS-BleoR was obtained through bleomycin resistance plate screening and colony PCR and sequencing verification. ...

Embodiment 3

[0145] The construction method of the Schizochytrium genetic engineering strain expressing EPA synthase gene of embodiment 3

[0146] This embodiment is a method for constructing a Schizochytrium genetically engineered strain expressing an EPA synthase gene, comprising the following steps in sequence:

[0147] S1. Construction of Recombinant Bacteria 1

[0148] S11. Preparation of Competent Agrobacterium AGL-1

[0149]Pick a single colony of Agrobacterium AGL-1 from the YEB solid medium containing 50 μg / mL carbenicillin (Carb) and transfer it to 5 mL of YEB liquid medium (containing 50 μg / mL Carb), at 28 ° C, 200 rpm In a shaker, cultivate for 12 hours;

[0150] Take 2 mL of the above-mentioned bacterial liquid into 50 mL of YEB liquid medium (containing 50 μg / mL Carb), and culture it in a shaker at 28 ° C and 200 rpm until the absorption value of the bacterial liquid at 600 nm is 0.5;

[0151] Take out the bacterial solution, after 30min in ice bath, centrifuge at 5000rpm ...

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Abstract

The invention discloses a schizochytrium limacinum genetic engineering strain for expressing an EPA (Eicosapentaenoic Acid) synthase gene and a construction method of the schizochytrium limacinum genetic engineering strain, and the schizochytrium limacinum genetic engineering strain is prepared by the following steps: by taking Schizochytrium sp.HX-308 as an original strain, inserting a Shewanella japonica EPA synthase encoding gene; the invention also discloses an application of the strain. The strain is used for producing EPA through fermentation. The engineering strain disclosed by the invention is good in stability, short in culture period, high in intracellular grease content and not limited by seasons, the synthesis amount of EPA is remarkably increased, and the obtained EPA is low in production cost and high in customer acceptance. The construction method is suitable for constructing the schizochytrium limacinum genetic engineering strain for expressing the EPA synthase gene, and the strain can be applied to biosynthesis of EPA.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a Schizochytrium genetically engineered strain, in particular to a Schizochytrium genetically engineered strain expressing an EPA synthase gene, a construction method and application thereof. Background technique [0002] Eicosapentaenoic acid (EPA) belongs to the omega-3 polyunsaturated fatty acid, and is a trace bioactive substance and an essential highly unsaturated fatty acid in the human body. EPA has the functions of lowering blood lipid, anti-tumor, anti-inflammation, anti-oxidative stress, stabilizing plaque, stabilizing cell membrane and reducing the risk of cardiovascular disease, etc. It is an important auxiliary treatment drug and health care product raw material. [0003] At present, the high-purity EPA on the market mainly comes from deep-sea fish oil, but extracting EPA from deep-sea fish oil has many disadvantages: [0004] (1) Restricted by season and place...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/80C12N9/10C12P7/6432C12R1/645
CPCC12N9/1029C12N15/80C12P7/6427
Inventor 黄和贾雨雷孙小曼农枋潼黄鹏伟马旺
Owner NANJING NORMAL UNIVERSITY
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