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Desaturase from arbuscular mycorrhizal fungi Delta 17 and application thereof

A saturase and unsaturated fatty acid technology, which is applied in applications, fungi, enzymes, etc., can solve the problems of limiting comprehensive catalytic activity and weak conversion rate

Active Publication Date: 2019-03-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The discovery and identification of these genes help to study the structure-function relationship of ω3Des, but their conversion rate to 18C substrates is weak, which limits their comprehensive catalytic activity to ω-6LC-PUFAs; The Δ17Des enzyme with improved comprehensive catalytic activity of ω-6LC-PUFAs has greater industrial application value for the transformation and optimization of fatty acid metabolism pathways to produce LC-PUFAs

Method used

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  • Desaturase from arbuscular mycorrhizal fungi Delta 17 and application thereof
  • Desaturase from arbuscular mycorrhizal fungi Delta 17 and application thereof
  • Desaturase from arbuscular mycorrhizal fungi Delta 17 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Determination of Δ17Des multiple sequence alignment template

[0026] Through literature research and sequence comparison of NCBI library, Δ17Des genes that currently prefer 20Cω-6LC-PUFAs were screened, and 6 genes were found to be reported (Saprolegniasis, Phytophthora infestans, Pythium melon, Sudden death of oak Phytophthora sojae, Phytophthora sojae, Phytophthora parasitica), and according to their catalytic efficiency to various substrates (see Table 1), Δ17Des (oPaFADS17) with the highest AA conversion rate was selected as the template.

[0027] Table 1 The reported catalytic efficiency of 20C-preferring Δ17Des on LA / GLA / DGLA and AA substrates

[0028]

Embodiment 2

[0029] Example 2: Δ17Des Multiple Sequence Alignment Determination of Candidate Genes

[0030] The gene sequence of Δ17 desaturase oPaFADS17, which prefers 20Cω-6LC-PUFAs, was used as a template for comparison in the NCBI library. According to the similarity and homology of the sequences, four gene sequences with high similarity and close species kinship were screened out. Combining with the host lipid content and fatty acid species of the research gene in the literature, the gene oRiFADS17 from a new species of arbuscular mycorrhizal fungi was finally determined as a candidate gene (the gene number is GBC46259.1, as shown in SEQ ID NO.2), NCBI The definition of this section of gene is acyl ω-3 fatty acid desaturase, fatty acid domain-containing protein, and endoplasmic reticulum enzyme, which are all characteristic of desaturase, but their specific functions are not described.

[0031] Using Clustal Omega software to compare the oRiFADS17 candidate gene with six 20C-preferrin...

Embodiment 3

[0034] Example 3: Construction and verification of recombinant expression vectors

[0035] 1. Codon optimization and synthesis of oRiFADS17 candidate genes

[0036] Using the sequence of oPaFADS17 as a template, the oRiFADS17 gene was determined by BLAST alignment. Because the codon preference of the gene sequence of arbuscular mycorrhizal fungi is different from that of the host Saccharomyces cerevisiae, in order to increase the expression of the target protein, we optimized the coding gene according to the codon usage of the natural sequence of the target strain, and synthesized oRiFADS17 Coding region, optimized gene sequence such as SEQ ID NO.3 (Nanjing GenScript Company, China). Their codon adaptation indices (CAI) were all raised to above 0.9 (CAI>0.8 is preferred). The synthetic sequence was ligated with the vector PUC57-simple through EcoRI and XhoI to obtain the subcloning vector PUC57-oRiFADS17, which was stored in E. coli Top 10.

[0037] 2. Construction of recom...

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Abstract

The invention discloses a desaturase from arbuscular mycorrhizal fungi Delta 17 and application thereof, and belongs to the technical field of biological engineering. The invention provides a gene forcoding delta 17Des. The nucleotide sequence is shown as SEQ ID NO.3. The gene is inserted into saccharomyces cerevisiae plasmid pYES2 / NT C; a positive converter is picked; sequencing verification iscompleted; then, a lithium acetate method is used for transferring the gene into saccharomyces cerevisiae INVSc1; the obtained recombinant strain is subjected to Western blot analysis; the protein expression quantity of the candidate delta17Des is studied; through exogenous addition of representative Omega-6LC-PUFAs substrates Al, GLA, DGLA and AA, activity verification is performed. The conditionthat the screened oRiFADS17 sequence has the delta17Des activity is proved; the application of enzyme coded by the gene in aspects of LC-PUFAs conversion is provided.

Description

technical field [0001] The invention relates to a Δ17 desaturase derived from arbuscular mycorrhizal fungus and application thereof, belonging to the technical field of bioengineering. Background technique [0002] Long-chain polyunsaturated fatty acids (LC-PUFAs) refer to straight-chain fatty acids containing two or more double bonds and having 20 or more carbon atoms, among which ω-3 PUFAs such as EPA (eicosapentaenoic acid, 20:5) And DHA (docosahexaenoic acid, 22:6), is an essential fatty acid for human health, and is essential for mammalian brain development, tissue formation and repair. Besides that, they also have remarkable effects in preventing asthma, obesity, depression, immune disorders, cardiovascular diseases and cancer. However, the natural sources of ω-3LC-PUFAs are limited, they cannot be synthesized in the human body, and they are mainly obtained from diet. At present, ω-3LC-PUFAs are mostly derived from deep-sea fish oil, but they have disadvantages such ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/64C12N15/53C12N15/63C12N1/19C12N1/21C12N1/15C12R1/66C12R1/19C12R1/84C12R1/865
CPCC12N9/0071C12P7/6427
Inventor 陈海琴唐鑫戎春驰赵建新张灏陈永泉陈卫
Owner JIANGNAN UNIV
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