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Recombinant yeast strain capable of producing conjugated linoleic acid and application thereof

A technology of conjugated linoleic acid and yeast, which is applied in the field of microbial production of conjugated linoleic acid, and can solve the problems of inability to use and low yield of conjugated linoleic acid

Active Publication Date: 2012-07-18
JIANGNAN UNIV
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  • Abstract
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  • Application Information

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Problems solved by technology

[0003] Although foreign researchers have expressed the linoleic acid isomerase gene PAI in tobacco seeds and rice, the yield of conjugated linoleic acid is very low, which is mainly due to the low oil content of these two plants, and with Unlike fatty acid dehydrogenase and elongase, the only form of PAI substrate is free fatty acid, and the form of phospholipid, methyl ester, Co-A or triglyceride of linoleic acid cannot be used as the substrate of PAI

Method used

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  • Recombinant yeast strain capable of producing conjugated linoleic acid and application thereof
  • Recombinant yeast strain capable of producing conjugated linoleic acid and application thereof
  • Recombinant yeast strain capable of producing conjugated linoleic acid and application thereof

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Embodiment 1

[0019] The codon optimization of embodiment 1 linoleic acid isomerase (PAI) gene

[0020] Referring to the pai gene sequence (genebank: AX062088), based on the characteristics of the Yarrowia lipolytica genome, the algorithm of the Genscript OptimumGeneTM system was used to optimize the design of pai transcription, translation, protein folding and other related parameters, including GC content, rare Codon usage, mRNA structure, and various cis elements during transcription and translation. At the same time, before the start codon of the gene sequence, a Kozak sequence that is conducive to the initiation of translation in eukaryotes: GCCACA is added. The opai nucleotide sequence after codon optimization is shown in SEQID NO: 1, and its codon adaptation index (CAI) has increased from 0.76 to 0.91. For specific results, see figure 1 . The optimized opai gene was synthesized by Nanjing GenScript Biotechnology Co., Ltd., and subcloned into the vector pUC57 (purchased from Nanjing...

Embodiment 2

[0021] Embodiment 2 Construction of recombinant expression plasmid

[0022] Plasmid pGEX6-1-PAI containing the natural linoleic acid isomerase gene pai (genebank: AX062088) derived from Propionibacterium acnes (Prof.Ivo Feussner, Georg-August-University, ttingen, Germany) as a template, with a pair of primers:

[0023] P1: 5′-CACGTGATGTCCATCTCGAAGG-3′ and

[0024] P2: 5′-GGTACCTTACACGAAGAACCGC-3′

[0025] The linoleic acid isomerase gene pai was amplified by PCR. The PCR program is: 15 seconds at 94°C, 30 seconds at 57°C, 1 minute at 68°C, 30 cycles. PCR reaction system: 5 μl 2mM dNTPs, 5 μl 10×Buf., 1 μl KOD plus, 2 μl 25 mM MgSO4, 1.5 μl upstream and downstream primers, 1 μl template. The amplified fragment was connected to the T vector pMD19T (purchased from TaKaRa Company) to obtain pMD19T-pai.

[0026] The subcloning vectors pUC57-opai and pMD19T-pai were digested with endonucleases Pml I and Kpn I at 37°C for 1.5h, separated and recovered by 1% agarose gel electropho...

Embodiment 3

[0027] Embodiment 3 Preparation of recombinant Yarrowia lipolytica

[0028] The three recombinant plasmids pINA1312-pai, pINA1312-opai and pINA1292-opai were digested with Not I at 37°C for 1.5h, separated and recovered by 1% agarose gel electrophoresis, and the expression unit fragment with the target gene was concentrated to 500ng / μl , Take 2μtg for transformation of Yarrowia lipolytica polh. The conversion process is as follows:

[0029] 1) Strain Y.lipolytica Polh was streaked on YPD solid medium, and cultured at 28°C for 12h;

[0030] 2) Pick a ring of Polh from the plate with an inoculation loop and resuspend in 1 mL TE solution;

[0031] 3) Centrifuge at 10,000 rpm to collect the bacteria, suspend the bacteria in 600 μl of 0.1 mol / l lithium acetate (pH6.0) buffer, bathe in 28°C water for 1 hour, and be careful not to shake the sample;

[0032] 4) After the water bath is over, centrifuge at 3000rpm for 2min, discard the supernatant;

[0033] 5) Gently resuspend the b...

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Abstract

The invention relates to a recombinant yeast strain capable of producing conjugated linoleic acid and an application thereof. In the invention, codon optimization is carried out on linoleate isomerase in propionibacterium acnes (PAI) gene to obtain an optimized gene opai, recombinant expression plasmid and recombinant Yarrowia lipolytica bacterium which contain the opai gene are prepared, and therecombinant Yarrowia lipolytica bacterium which expresses the opai gene is utilized to produce conjugated linoleic acid.

Description

technical field [0001] The present invention relates to the microbial production of conjugated linoleic acid, and specifically provides a recombinant yeast strain for producing conjugated linoleic acid, a preparation method of the strain and the use of the strain in the production of conjugated linoleic acid. Background technique [0002] Conjugated linoleic acid (CLA) is a general term for octadecadienoic acid mixtures with conjugated double bonds. In the past three decades, CLA has received extensive attention due to its diverse physiological functions. The main physiological functions of CLA include anti-cancer, anti-atherosclerosis, inhibition of fat accumulation, promotion of growth, and stimulation of immunity. Among them, c9, t11-CLA, t10, c12-CLA and t9, t11CLA are three conjugated linoleic acid monomers confirmed to have physiological activity so far. Different monomers have different physiological activities and signal transduction mechanisms. In nature, CLA is p...

Claims

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Application Information

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IPC IPC(8): C12N15/61C12N15/63C12N1/19C12P7/64C12R1/645
Inventor 张灏宋元达张白曦陈卫陈海琴刘晓明赵建新
Owner JIANGNAN UNIV
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