Recombinant yeast strain producing nervonic acid and its application

A yeast strain and yeast technology, applied in the field of engineered recombinant yeast strains, can solve the problems of wild strains being unable to synthesize nervonic acid, difficult to meet industrial needs, and shortages, and achieve good industrial application prospects and the effect of increased production

Active Publication Date: 2021-07-27
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the fatty acids in Y. lipolytica are C16 and C18 fatty acids. Due to the lack of carbon chain elongase and fatty acid desaturase necessary for the synthesis of ultra-long chain monounsaturated fatty acids, wild strains cannot synthesize nervonic acid.
Qiao Jianwen introduced fatty acid elongase (AtFAE1, BtFAE1 and CgKCS), desaturase (SCD) and diglyceride acyltransferase (DGAT1) into Yarrowia lipolytica through genetic engineering, and the constructed recombinant yeast cells can produce Nervous acid, but its content only accounts for 1.5% of the total oil content in cells, which is difficult to meet industrial needs

Method used

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  • Recombinant yeast strain producing nervonic acid and its application
  • Recombinant yeast strain producing nervonic acid and its application
  • Recombinant yeast strain producing nervonic acid and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0127] Embodiment 1, plasmid construction

[0128] 1.1 Cloning of genetic elements

[0129] 1) Acquisition of genes DGAT1, SCD, PLA2-1, PLA2-2, PLA2-3, PLA2-4, PLA2-5, PLA2-6, ylGSR and ylGPO:

[0130] Yarrowia lipolytica strain (strain number polg, purchased from Yeastern Biotech Company, Taiwan) in YPD medium (YPD medium composition is glucose 20g / L, peptone 20g / L, yeast extract 10g / L)) After culturing, high-purity total genomic DNA was extracted by the CTAB (hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide) method. Add appropriate amount of bacteria into liquid nitrogen to freeze, grind into powder, add appropriate amount of 2×CTAB extraction buffer (100mmol / L Tris-HCl, pH8.0, 20mmol / L EDTA, 1.4mol / L NaCl, 2% (w / v )CTAB, 40mmol / L mercaptoethanol), at 65°C for 10 minutes, with intermittent shaking. Then add an equal volume of chloroform / isoamyl alcohol, gently invert the centrifuge tube to mix, centrifuge at 12,000 rpm for 10 min at room temperature,...

Embodiment 2

[0158] Embodiment 2, the construction of Yarrowia lipolytica engineering bacteria

[0159] 2.1 Obtaining the expression cassette

[0160] Plasmids pDS, pAB, pCgKCS, and pCgKCS recorded in Table 1 were digested with NotI endonuclease (purchased from Thermo Fisher Scientific) ER pCgKCS PTS , pCgKCS MTS , pC ER C MTS , pMCSD, pCB, pPLA2-1, pPLA2-2, pPLA2-3, pPLA2-4, pPLA2-5, pPLA2-6, pylGSR, pylGPO, prELO2, pCpLCE, pgELOVL6, pMaKCS, pD9DMB, pCeFAT6, pMaOLE2, pAtADS1, pAtADS2 , pEcAldH, pScZwf and pΔpex10.

[0161] The specific digestion system is: 10×FD Green Buffer, 2ul; NotI, 1ul; plasmid, 2 O make up to 20ul. The digested product was purified and recovered using Cycle Pure Kit (purchased from OMEGA bio-tek). The recovery steps were as follows: add 4-5 times the volume of buffer CP to the digested product; transfer to a DNA adsorption column after mixing, and centrifuge at 13,000g at room temperature for 1 Discard the filtrate and add 700 μL DNA washing buffer, centrifu...

Embodiment 3

[0193] Embodiment 3, bacterial strain culture produces nervonic acid

[0194] 3.1 Strain shake flask culture and induction regulation

[0195] a. Activate the strains po1g and YL2-3 on the YPD solid plate, and culture them at 28°C for 1 day. Pick single colonies and inoculate them into 250ml shake flasks filled with 50ml YPD medium respectively, and cultivate them at 28°C for 1 day as the seed culture solution. Inoculate the seed culture solution into 250ml shake flasks containing 50ml YNB medium respectively to make the initial OD 600 0.2, cultured at 28°C for 6 days, ready to use.

[0196] Among them, the composition of YNB medium is 1.7g / L of YNB, 80g / L of glucose, 1.5g / L of yeast extract, 20mg / L of uracil, and 100mg / L of leucine.

[0197] b. The seed culture solution cultivated by the above method was inoculated into 250ml shake flasks containing 50ml induction medium respectively to make the initial OD 600 0.2, cultured at 28°C for 6 days, ready to use.

[0198] Wher...

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Abstract

The invention discloses an engineered yeast strain producing nervonic acid. The yeast strain overexpresses genes related to enzymes required for the synthesis of long-chain unsaturated fatty acids such as fatty acid elongase, desaturase, and diglyceride acyltransferase, and any Optionally, the triglyceride synthesis and decomposition pathway, the sphingomyelin synthesis and decomposition pathway, the lipid subcellular level synthesis and decomposition pathway, and the redox balance pathway of the strain are further regulated. The recombinant yeast strain can produce microbial oil, and the content of the prepared nervonic acid accounts for 39.6% of the total fatty acid content.

Description

technical field [0001] The invention belongs to the field of biotechnology. More specifically, the present invention relates to an engineered recombinant yeast strain capable of efficiently producing nervonic acid (cis-15-tetracosanemonoic acid, also known as squalic acid, C24:1, Δ15) at a high concentration. Background technique [0002] Unsaturated fatty acids are mostly essential fatty acids for the human body, which can regulate blood lipids, clear blood clots, nourish the brain, relieve inflammation, etc., mainly including monounsaturated fatty acids and polyunsaturated fatty acids. Among them, very long chain monounsaturated fatty acid (VLCMFA) is an unsaturated fatty acid with more than 18 carbon atoms in the main carbon chain and only one double bond. The common one is cod oleic acid (Eicosenoic acid , C20:1Δ11), erucic acid (C22:1Δ13), nervonic acid (C24:1Δ15) and Ximenynic acid (C26:1Δ17). Ultra-long-chain monounsaturated fatty acids have unique medicinal effects...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12P7/64
CPCC12N9/0071C12N9/1029C12N9/0008C12N9/0006C12N9/0051C12N9/0065C12P7/6463C12P7/6409C12Y114/19001C12Y101/01049C12Y108/01007C12Y111/01009C12P7/64C12Y114/99C12Y203/01199C12Y203/0102C12N15/815C12N1/16C12N9/18C12Y102/01003C12Y301/01004
Inventor 孟慧敏王士安李家欣李福利
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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