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Yarrowia lipolytica genetically engineered bacteria and application thereof

A technology of Yarrowia lipolytica and genetically engineered bacteria, which is applied in the field of genetic engineering and can solve the problem of low yield

Inactive Publication Date: 2020-11-24
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, wax esters can be produced in Escherichia coli and Saccharomyces cerevisiae by exogenously introducing genes related to the wax ester synthesis pathway, but the yields are relatively low without adding additional precursors

Method used

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  • Yarrowia lipolytica genetically engineered bacteria and application thereof
  • Yarrowia lipolytica genetically engineered bacteria and application thereof
  • Yarrowia lipolytica genetically engineered bacteria and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1 Construction of Yarrowia lipolytica genetically engineered bacteria producing very long-chain fatty acids (VLCFAs)

[0093] (1) Construct the elongase gene AtKCS, CraKCS and MaELO3 expression plasmids p32UTAtKCS, p32UTCraKCS and p32UTMaELO3 with UAS4B+TEF promoters respectively.

[0094] Using primers 32UTAtKCS-f and 32UTAtKCS-r (the nucleotide sequences of which are respectively shown in the sequence table SEQ ID NO: 1-2) to extract the optimized sequence of the elongase gene AtKCS (its nucleus from Arabidopsis thaliana) The nucleotide sequence is shown in SEQ ID NO: 23) was constructed into the plasmid p3204 whose promoter is UAS4B+TEF through restriction sites Pmll and BamHI to obtain plasmid p32UTAtKCS. Using primers 32UTCraKCS-f and 32UTCraKCS-r (the nucleotide sequences thereof are respectively shown in the sequence table SEQ ID NO: 3-4) to extract the optimized sequence (the nucleotide The sequence is shown in SEQ ID NO: 24) was constructed into the pl...

Embodiment 2

[0104] Embodiment 2 Yarrowia lipolytica genetically engineered bacteria produce the determination of the ratio of each chain length fatty acid

[0105] The bacterial strain Yarrowia lipolytica Po1f, the bacterial strain GQ03 prepared in Example 1, GQ05, GQ07 were respectively inoculated in 2mLYPD medium (this YPD medium is made up of 2% glucose, 2% peptone and 1% yeast extract, and the balance is water, said percentage is mass percent), cultured for 24 hours, then with initial OD 600 The inoculum of 0.01 was inoculated into new 50mL YPD medium for culture. After 3 days of fermentation and cultivation, fatty acids were extracted and then derivatized into fatty acid methyl esters, and the ratio of each fatty acid methyl ester was detected by GC (gas chromatography).

[0106] Extraction of fatty acids: After the fermentation, take 20 mL of the fermentation broth into a centrifuge tube, centrifuge at 4500 rpm for 5 minutes, resuspend with 20 mL of deionized water and wash twice. ...

Embodiment 3

[0113] Example 3 Construction of Yarrowia lipolytica genetically engineered bacteria producing ultra-long-chain wax esters

[0114] (1) The expression plasmids p32UTMaFAR and p32UTTaFAR of fatty acyl-CoA reductase genes MaFAR and TaFAR with UAS4B+TEF promoters were respectively constructed.

[0115] The optimization of the fatty acyl-CoA reductase gene MaFAR derived from Marinobacter aquaeolei VT8 Maqu_2220 was performed using primers 32UTMaFAR-f and 32UTMaFAR-r (the nucleotide sequences of which are shown in the sequence table as SEQ ID NO: 13-14, respectively). The sequence (the nucleotide sequence of which is shown in SEQ ID NO: 26) was constructed into the plasmid p3204 with the promoter UAS4B+TEF through the restriction sites Pmll and BamHI to obtain the plasmid p32UTMaFAR. Use primers 32UTTaFAR-f and 32UTTaFAR-r (the nucleotide sequences of which are respectively shown in the sequence table as SEQ ID NO: 15-16) to extract the fatty acyl-CoA reductase derived from barn ow...

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Abstract

The invention provides a yarrowia lipolytica genetically engineered bacteria producing wax esters. The yarrowia lipolytica genetically engineered bacteria producing wax esters is obtained by overexpressing wax ester synthase and fatty acyl coenzyme A reductase gene based on yarrowia lipolytica engineering bacterium capable of producing ultra-long-chain fatty acids. The yarrowia lipolytica engineering bacterium capable of producing ultra-long-chain fatty acids is constructed by knocking an AtKCS gene, a CraKCS gene and an MaELO3 gene into a chromosome of yarrowia lipolytica which knocked out aPEX10 gene. Finally, four yarrowia lipolytica engineering bacteria WE01, WE02, WE03, WE04 which produc wax esters are constructed, wherein the WE01 engineering bacteria can be fermented through continuous feeding, and the yield of the wax esters can achieve 2g / L. The constructed yarrowia lipolytica genetically engineered bacteria is simple to operate and reliable in performance, and can be appliedto large-scale commercial production.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a Yarrowia lipolytica genetically engineered bacterium and its application. Background technique [0002] Wax esters are neutral lipids formed by the esterification of long-chain fatty alcohols and long-chain fatty acids. It widely exists in animals, plants and microorganisms in nature, and is of great significance to their life activities. Due to its excellent properties, wax esters are widely used in various fields such as cosmetics, medicine and food. Since the World Whaling Convention banned the fishing of sperm whales, liquid wax esters derived from jojoba (Simmondsia chinensis oil) have become the main source of commercial wax esters, and the market demand continues to grow. Compared with other wax esters, it shows higher the value of. Jojoba wax ester is a mixture of super long-chain wax esters. It is composed of 39 different wax esters. The main wax ester...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/53C12N15/54C12P7/64C12R1/645
CPCC12N9/1029C12N9/0008C12Y102/0105C12Y203/01199C12Y203/01075C12N15/52C12N15/815C12P7/6436C12P7/6409
Inventor 花强韦柳静高琪杨敬林赵鑫茹陈骏
Owner EAST CHINA UNIV OF SCI & TECH
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