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Recombinant mortierella alpina strain for heterologous expression of MpFADS6 gene, construction method of recombinant mortierella alpina strain and application of recombinant mortierella alpina strain in production of EPA

A kind of Mortierella alpine, heterologous expression technology, applied in the field of bioengineering

Active Publication Date: 2016-02-03
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that in order to increase the EPA production of Mortierella alpina, the main idea adopted in the prior art is to increase the EPA production by consuming AA, and to change the fatty acid synthesis pathway of Mortierella alpina through the ω3 pathway (ALA → EPA), and to consume ALA to increase the EPA production. Research on improving EPA production has not been reported

Method used

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  • Recombinant mortierella alpina strain for heterologous expression of MpFADS6 gene, construction method of recombinant mortierella alpina strain and application of recombinant mortierella alpina strain in production of EPA
  • Recombinant mortierella alpina strain for heterologous expression of MpFADS6 gene, construction method of recombinant mortierella alpina strain and application of recombinant mortierella alpina strain in production of EPA
  • Recombinant mortierella alpina strain for heterologous expression of MpFADS6 gene, construction method of recombinant mortierella alpina strain and application of recombinant mortierella alpina strain in production of EPA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Synthesis of FADS6 gene (MpFADS6) derived from Micromonas pumilus CCMP1545

[0060] According to the sequence information of the MpFADS6 gene derived from Micromonas pumilus CCMP1545 (GenBank number KU236373), primers P1 and P2 were designed, the underlines were the enzyme cutting sites EcoRI and SmaI respectively, and the plasmid PUC-MpFADS6 containing the MpFADS6 gene was used as a template (Shanghai Sunny Biotechnology Co., Ltd.), P1 / P2 as primers, using KOD high-fidelity polymerase to obtain the MpFADS6 fragment by PCR. The PCR program was: 94°C for 40s, 55°C for 40s, 68°C for 1.5min, 30 cycles, and the PCR amplified products were separated by 1.0% agarose gel electrophoresis and recovered.

[0061] P1 (sense): 5'-TACCG GAATTC ATGTGCCCGCCGAAGACGGACGGC-3'

[0062] P2 (antisense): 5'-TCTAG CCCGGG TCAGTGCGCCTTTCTCCGCCTTGCC-3'

Embodiment 2

[0063] Example 2: Construction of binary expression vector pBIG2-ura5s-MpFADS6

[0064] 1. Enzyme digestion reaction

[0065]At 37°C, the MpFADS6 fragment and the vector pBIG2-ura5s-ITs fragment were digested with restriction enzymes EcoRI and SmaI, the product was purified to recover the MpFADS6 fragment, and the pBIG2-ura5s fragment was recovered by gel cutting and purification. Enzyme digestion system (50μL): 1μL EcoRI (Cat. No. FD0275), 1 μL SmaI (Cat. No. FD0663), 40 μL plasmid or PCR product, 5 μL 10X Buffer (Fermentas Company), 3 μL of deionized water, incubated at 37°C for 2h.

[0066] 2. Ligation reaction

[0067] The digested MpFADS6 fragment MpFADS6 was ligated with the vector pBIG2-ura5s using T4 ligase, and incubated at 4°C for 12 hours to obtain the recombinant expression vector pBIG2-ura5s-MpFADS6. The ligation system is (10 μL): 5 μL of the digested fragment of the target gene, 3 μL of the digested fragment of the vector, 1 μL of 10X ligase buffer, 1 μL...

Embodiment 3

[0080] Example 3: Agrobacterium tumefaciens-mediated transformation of Mortierella alpina strain MAU1 (CCFM501, namely CGMCCNo.8414)

[0081] On the basis of the existing domestic and foreign literature reports on the transformation method of Agrobacterium tumefaciens, appropriate optimization was carried out, as follows:

[0082] 1) Agrobacterium tumefaciens C58C1 containing the pBIG2-ura5s-MpFADS6 plasmid stored at -80°C were streaked on YEP solid medium plates containing 100 μg / mL rifampicin and 100 μg / mL kanamycin. Incubate in the dark at 28°C for 48 hours.

[0083] 2) Pick a single clone and inoculate it into 15 mL of liquid YEP medium containing 100 μg / mL rifampicin and 100 μg / mL kanamycin at 28° C., 200 rpm, and incubate in the dark for 36 hours.

[0084] 3) Centrifuge at 8000g for 5 minutes to collect the bacterial cells, and discard the supernatant. Add 5mL IM medium to resuspend the bacteria, centrifuge at 8000g for 5 minutes, and discard the supernatant. Add 2mL ...

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Abstract

The invention provides a recombinant mortierella alpina strain for heterologous expression of an MpFADS6 gene by virtue of a delta-6 desaturase gene MpFADS6 from tiny microcystis. The invention also provides an application of the recombinant mortierella alpina strain in production of EPA. By adding 1% of peony seed oil or 50g / L of peony seed pulp juice into a culture medium, the yield of the EPA is effectively increased, which has important significance for basic theory research of metabolic regulation in synthesis of fatty acid of an oil-producing fungus namely mortierella alpine and production of PUFAs. Compared with over-expression of an omega 3 fatty acid desaturase gene in the mortierella alpine, the yield of the EPA is increased by 5 times and can reach 588.5+ / -29.6mg / L.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and the invention relates to a Mortierella alpina engineering strain heterologously expressing the MpFADS6 gene, in particular to a recombinant Mortierella alpina strain heterologously expressing the MpFADS6 gene derived from Micromonas micromonas CCMP1545, and also relates to Its construction method, and its use in the production of EPA. Background technique [0002] Eicosapentaenoic acid (eicosapentaenoic acid, EPA, 20:5 Δ5,8,11,14,17 ) is a common omega-3 polyunsaturated fatty acid (Polyunsaturatedfattyacid, PUFAs). PUFAs such as EPA have physiological functions such as anti-cancer, anti-atherosclerosis, and enhancing the body's immunity, which are of great significance to maintaining health. The dietary supplementation of polyunsaturated fatty acids has been clearly written into the dietary reference guidelines of the United States and the United Kingdom. my country also recommends a...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/80C12P7/64C12R1/645
Inventor 陈海琴陈永泉史海粟顾震南张灏陈卫
Owner JIANGNAN UNIV
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