Unlock instant, AI-driven research and patent intelligence for your innovation.

A δ4 fatty acid desaturase derived from Euglena microsecta and its application

A desaturase and fatty acid technology, applied in the field of Δ4 fatty acid desaturase, can solve problems such as difficult synthesis, complex synthesis, and inability to increase DHA

Inactive Publication Date: 2016-02-10
JIAMUSI UNIVERSITY
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, when the fat-1 gene is used to increase the content of long-chain polyunsaturated fatty acids in mammals, the key deficiency is that it cannot increase the content of DHA (22:6n-3)
[0005] In fact, the synthesis of DHA (22:6n-3) in mammals is very complex, through a so-called "Sprecher pathway", EPA (20:5n-3) is elongated to generate an intermediate product DPA (22:5n-3 ), and then further extended to 24:5n-3, and then quickly generated 24:6n-3 by △6 desaturase, and then further produced DHA (22:6n-3) by β-oxidation, this complex Mechanism may be responsible for mammals' difficulty in synthesizing high levels of DHA (22:6n-3)

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A δ4 fatty acid desaturase derived from Euglena microsecta and its application
  • A δ4 fatty acid desaturase derived from Euglena microsecta and its application
  • A δ4 fatty acid desaturase derived from Euglena microsecta and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1. Synthesis and functional verification of Δ4 fatty acid desaturase gene (sEgD4 gene)

[0056] 1. Synthesis of Δ4 fatty acid desaturase gene (sEgD4 gene)

[0057] Codon optimization was performed on the cDNA sequence (the full length of the coding frame is 1626bp) of the Δ4 fatty acid desaturase gene (GenBank number: AY278558) of Euglenagracilis submitted in GenBank to make it conform to the codon usage preference of mammalian genes . After optimization, use software to predict the secondary structure of its mRNA to verify whether the codon optimization is reasonable.

[0058] The nucleotide sequence of the optimized Δ4 fatty acid desaturase gene (sEgD4 gene) is shown in SEQIDNo.1 from the 10th to the 1635th nucleotide from the 5' end, and the encoded amino acid sequence is shown in SEQIDNo.2 shown.

[0059] The optimized sequence was added with EcoRI and HindIII restriction sites at both ends, and then the full-length gene was artificially synthesized. The ...

Embodiment 2

[0090] Example 2. Synergy between Δ4 fatty acid desaturase encoded by sEgD4 gene and Δ15 fatty acid desaturase in mammalian cells

[0091] 1. According to research reports, the expression product of the fat-1 gene (GenBank number: NM_001028389) from the nematode C. elegans has Δ15 fatty acid desaturase activity, and the codon-optimized design of the fat-1 gene was artificially synthesized As shown in SEQ ID No.3.

[0092] The coding gene sequence of the Δ15 fatty acid desaturase is shown in SEQ ID No.3 from the 13th to the 1221st nucleotide from the 5' end, and the amino acid sequence of the Δ15 fatty acid desaturase is shown in SEQ ID No.4.

[0093] The recombinant expression plasmid pcDNA3.1-sD15 was obtained according to the method in Step 2 of Example 1.

[0094] 2. According to the cell transfection method in step 3 in Example 1, the expression plasmids pcDNA3.1-EGFP, pcDNA3.1-sD15, pcDNA3.1-sD15+pcDNA3.1-sEgD4 were transfected and plated in CHO cells on a 60mm plate , ...

Embodiment 3

[0101] Example 3. Synergy between Δ4 fatty acid desaturase encoded by sEgD4 gene and Δ6 / Δ5 fatty acid desaturase in mammalian cells

[0102] pcDNA3.1-F2F1 is a double-gene expression vector containing mouse-derived Δ6-fatty acid desaturase gene fads2 (GenBank No.: BC057189) and Δ5-fatty acid desaturase gene fads1 (GenBank No.: BC063053).

[0103] The coding gene sequence of Δ6-fatty acid desaturase is shown in SEQ ID No.5, and the amino acid sequence of the protein is shown in SEQ ID No.6.

[0104] The coding gene sequence of Δ5-fatty acid desaturase is shown in SEQ ID No.7, and the amino acid sequence of the protein is shown in SEQ ID No.8.

[0105] According to the cell transfection method in Step 3 in Example 1, the expression plasmids pcDNA3.1-EGFP, pcDNA3.1-F2F1, pcDNA3.1-F2F1+pcDNA3.1-sEgD4 were transfected and plated in CHO cells on a 60mm plate, and each The plasmids used in each transfection group were 8 μg (4 μg for each of the two plasmids in pcDNA3.1-F2F1+pcDNA3.1...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses Delta4 fatty acid desaturase from euglena gracilis and the application of the Delta4 fatty acid desaturase. The invention discloses (1) a protein shown in SEQ ID No. 2, or (2) proteins with the same function, obtained by replacing and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 2. The Delta4 fatty acid desaturase can be used for promoting the production of important long-chain polyunsaturated fatty acid such as high-level docosahexaenoic acid in mammals, and can also be used for converting DPA (docosapentaenoic acid) produced by various methods into DHA in mammalian cells or mammals.

Description

technical field [0001] The invention relates to a Δ4 fatty acid desaturase derived from Euglena microsecta and its application. Background technique [0002] Fatty acids are divided into saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids (PUFAs). Among them, PUFAs have more unique effects and significance on the human body, and are a class of fatty acids that have been extensively studied and received much attention. PUFAs include two types of ω-3 and ω-6 PUFAs, and each type includes multiple members with different carbon chain lengths and different numbers of unsaturated bonds. PUFAs with a carbon chain length ≥ 20 are called long-chain polyunsaturated fatty acids (LCPUFAs), and the most important LCPUFAs include DHA (22:6n-3), EPA (20:5n-3) and ARA (20:4n-6 , that is, arachidonic acid), they are the three most biologically active unsaturated fatty acids in organisms, which have been proven to promote brain development and function mainten...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/85C12N5/10C12P7/64
CPCC12N9/0071C12N15/85
Inventor 朱贵明王淑秋孙洁王迪迪葛堂栋江旭东朴金花
Owner JIAMUSI UNIVERSITY