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Recombinant mortierella alpina strain for heterologous expression of linoleic acid isomerase gene and construction method thereof

A technology of linoleic acid isomerase and Mortierella alpina, applied in the field of bioengineering, can solve problems such as changing the lipid metabolism pathway of Mortierella alpina

Inactive Publication Date: 2015-05-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there has been no report of changing the lipid metabolism pathway of Mortierella alpina by introducing exogenous genes, so that Mortierella alpina can produce exogenous functional fatty acid species

Method used

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  • Recombinant mortierella alpina strain for heterologous expression of linoleic acid isomerase gene and construction method thereof
  • Recombinant mortierella alpina strain for heterologous expression of linoleic acid isomerase gene and construction method thereof
  • Recombinant mortierella alpina strain for heterologous expression of linoleic acid isomerase gene and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Cloning of the linoleic acid isomerase gene (PAI) derived from Propionibacterium acnes and artificial synthesis of the codon-optimized gene (oPAI)

[0056] According to the sequence information of the PAI gene derived from Propionibacterium acnes, primers P1 and P2 were designed, the underlines were the restriction sites HindⅢ and SacI respectively, and the plasmid PMD19T-PAI containing the linoleic acid isomerase gene (PAI) was used as the template ( Baixi Zhang, Chunchi Rong, Haiqin Chen, Yuananda Song, Hao Zhang, Wei Chen. De novosynthesis of trans-10, cis-12 conjugated linoleic acid in oleaginous yeast Yarrowia Lipolytica. Microbial Cell Factories. 2012, 11), with primers P1 / P2 , using KOD high-fidelity polymerase to obtain PAI fragments by PCR. The PCR program was: 94°C for 30s, 55°C for 30s, 68°C for 1.5min, 30 cycles, and the PCR product was purified, and the purified product was verified by 1.0% agarose gel electrophoresis.

[0057] P1 (sense): TACCC...

Embodiment 2

[0060] Example 2: Construction of binary expression vectors pBIG2-ura5s-PAI and pBIG2-ura5s-oPAI

[0061] 1. Enzyme digestion reaction

[0062] Under the condition of 37°C, the PCR purified product of the linoleic acid isomerase fragment PAI, the plasmid pUC19-oPAI and the vector pBIG2-ura5s-ITs fragment were cut with restriction endonuclease HindⅢ, and the target DNA was purified and recovered. The HindⅢ digestion system (100 μL) was: 2 μL HindⅢ-HF, 30 μL plasmid or PCR product, 10 μL Buffer2, 58 μL deionized water, and incubated at 37°C for 12 hours.

[0063] The target DNA was further digested with restriction endonuclease SacI, purified and recovered by gel cutting. Enzyme digestion system (100 μL): 2 μL SacI, 30 μL plasmid or PCR product, 10 μL Buffer4, 58 μL deionized water, enzyme digestion in 37°C water bath for 12 hours.

[0064] The composition of Buffer 2 endonuclease damping solution involved in the present invention is as follows: 50mM NaCl, 10mM Tris-HCl, 10mM ...

Embodiment 3

[0078] Example 3: Agrobacterium tumefaciens-mediated transformation of Mortierella alpina strain MAU1 (CCFM501, CGMCC No.8414)

[0079] On the basis of the existing domestic and foreign literature reports on the transformation method of Agrobacterium tumefaciens, appropriate optimization has been carried out. The specific successful implementation examples are as follows:

[0080] 1) Streak Agrobacterium tumefaciens C58C1 containing plasmids pBIG2-ura5s-PAI and pBIG2-ura5s-oPAI stored at -80°C on YEP solid culture containing 100 μg / mL rifampicin and 100 μg / mL kanamycin, respectively base plate. Incubate in the dark at 28°C for 48 hours.

[0081] 2) Pick a single clone and inoculate it into 20 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.

[0082] 3) Centrifuge at 4000g for 5 minutes to collect the bacterial cells, and discard the supernatant. Add 5mL IM medium to resuspend the bacte...

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Abstract

The invention provides recombinant mortierella alpina oPAI for heterologous expression of linoleic acid isomerase gene from propionibacterium acnes and provides a method for constructing a recombinant mortierella alpina strain for heterologous expression of linoleic acid isomerase gene from propionibacterium acnes, and the invention further provides a use of the recombinant mortierella alpina strain for producing fatty acid, namely biological synthesizing conjugated linoleic acid. With mortierella alpina uracil auxotroph strain MAU1 as a material, a recombinant strain for heterologous expression of linoleic acid isomerase gene from propionibacterium acnes is successfully constructed, and a target product conjugated linoleic acid is detected in the recombinant strain added with long-chain acyl-coenzyme A for synthesizing an enzyme inhibitor.

Description

technical field [0001] The invention relates to an engineering strain heterologously expressing the linoleic acid isomerase gene in Mortierella alpina, especially a recombinant Mortierella alpina strain heterologously expressing the linoleic acid isomerase gene derived from Propionibacterium acnes The construction method thereof belongs to the technical field of bioengineering. Background technique [0002] Conjugated linoleic acid (CLA) is a class of octadecadienoic acid containing conjugated double bonds, and the two most important active monomers are cis-9, trans-11 CLA and trans- 10. cis-12 CLA, all have physiological functions such as anti-cancer, anti-atherosclerosis, and enhancing immunity of the body. Among them, trans-10 and cis-12CLA can also reduce the accumulation of body fat, so as to achieve the effect of weight loss. These two conjugated linoleic acids have passed the GRAS certification of the US FAD and can be used as safe food additives. However, the trad...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/80C12P7/64C12R1/645
CPCC12N9/90C12P7/6427C12Y502/01005
Inventor 陈海琴陈卫陈永泉郝丹辉郝光飞杨波张白曦顾震南张灏
Owner JIANGNAN UNIV
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