Recombinant strain for expressing heterogenous Omega-3 desaturase in mortierella alpina and construction method thereof

A technology of Mortierella alpina and saturase, which is applied in the field of bioengineering and can solve the problems of unreachable EPA production

Inactive Publication Date: 2016-05-11
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Yet this EPA output still can't reach the expectation of those skilled in the art under normal temperature environment

Method used

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  • Recombinant strain for expressing heterogenous Omega-3 desaturase in mortierella alpina and construction method thereof
  • Recombinant strain for expressing heterogenous Omega-3 desaturase in mortierella alpina and construction method thereof
  • Recombinant strain for expressing heterogenous Omega-3 desaturase in mortierella alpina and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Codon optimization and artificial synthesis of ω-3 desaturase gene derived from Pythium melon

[0048] Since the codon usage preference of the natural PaD17 gene sequence is different from that of the host Mortierella alpina, according to the codon usage preference of Mortierella alpina, the nucleic acid sequence of PaD17 was codon-optimized by GenscriptOptimumGeneTMsystem, and the optimized sequence was synthesized artificially. The latter gene sequence oPaFADS17, as shown in SEQNo.1 and 2, has added HindIII and XhoI restriction sites on both sides, and is connected with the PUC57 carrier to obtain PUC57-oPaFADS17, which is stored in Escherichia coli Top10 (GenScript, Nanjing, China) ,China). The codon availability index and codon usage frequency distribution of genes before and after optimization are as follows: figure 1 shown. In the figure, when CAI is 1.0, it means perfect in the desired expression system, and >0.89 means excellent, with high gene expr...

Embodiment 2

[0049] Example 2: Enzymatic cleavage reaction

[0050] At 37°C, use the restriction enzyme HindIII to digest the plasmid PUC57-oPaFADS17 and the vector pBIG2-ura5s-ITs fragment overnight with the restriction enzyme HindIII. 58 μL of deionized water, incubate at 37°C for 12h.

[0051]Among them, the vector pBIG2-ura5s-Its is directly obtained according to the content disclosed in Chinese patent application CN201310524221.4. The HPH expression unit was obtained from the pD4 plasmid by PCR, digested with the restriction enzymes EcoRI and XbaI, and inserted into the multiple cloning site (MCS) of pET28a(+) cut by EcoRI and XbaI. , the plasmid pET28a-HPHs was obtained. The ura5 (orotate phosphoribosyltransferase; OPRTase) gene was obtained from the cDNA of Mortierella alpina by PCR, and the ura5 gene was digested with the restriction enzymes BspHI and BamHI, and the digested ura5 gene was inserted into NcoI and BamHI. The plasmid pET28a-HPHs digested with BamHI was replaced with...

Embodiment 3

[0054] Example 3: Ligation reaction

[0055] The purified ω-3 desaturase gene fragment oPaFADS17 was ligated with the vector pBIG2-ura5s-ITs with T4 ligase, and ligated at 4°C for 12 h to obtain the recombinant expression vector pBIG2-ura5s-oPaFADS17. The ligation system was (10 μL): 2 μL of the target gene digested fragment, 3 μL of the vector digested fragment, 1 μL of ligase buffer, 1 μL of T4 ligase, 3 μL of sterile water, and ligated at 4°C for 12 h.

[0056] The ligation product was transformed into E. coli TOP10 competent cells, and the transformation method was as follows:

[0057] (1) Take 100 μL of competent cells under sterile conditions, add 1-2 μL of ligation product, and mix by pipetting.

[0058] (2) Transfer the mixed competent cells into the pre-cooled rotor cup to avoid bubbles.

[0059] (3) Put the electroporation cup into the Bio-Rad electroporator, adjust to the appropriate preset program gear, and electroporate. The voltage condition is 1.8kv.

[0060...

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Abstract

The invention provides a recombinant mortierella alpina strain Ma-oPaFADS17 with heterologous expression coming from pythium aphanidermatum Omega-3 desaturase genes and a method for constructing the mortierella alpina strain with heterologous expression coming from pythium aphanidermatum Omega-3 desaturase genes. The method adopts mortierella alpine uracil auxotrophic strain CCFM 501 as materials and uses an agrobacterium tumefaciens mediated genetic manipulation technology to obtain a recombinant strain with a high yield of EPA in the environment of normal temperature, wherein the yield of EPA reaches 617.1 mg / L, and the content of the EPA accounts for 18.7 percent of the total amount of fatty acid, which has an important meaning for the basic research and product development of oil-producing fungus mortierella alpine.

Description

【Technical field】 [0001] The invention relates to a genetically engineered strain in Mortierella alpine that overexpresses an ω-3 desaturase oPaFADS17 derived from Pythium citrullus and a construction method thereof, belonging to the technical field of bioengineering. 【Background technique】 [0002] Mortierella alpina is an oil-producing fungus that can accumulate lipids up to 50% of the dry cell weight, and is an important model organism for basic research on lipid biochemistry. Mortierella alpina has been used in the industrial production of arachidonic acid (Arachidonic acid, AA, C20:4), and the edible oils and fats it produces have passed the safety assessment of the United States Department of Agriculture (FDA). In addition to synthesizing AA, Mortierella alpina also has a certain ability to synthesize eicosapentanoic acid (EPA, C20:5). EPA belongs to omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) and has important physiological functions, such as: the abili...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/80C12P7/64C12R1/645
CPCC12N9/0071C12P7/6427
Inventor 陈海琴陈永泉陈卫梅甜甜顾震南张灏
Owner JIANGNAN UNIV
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