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Recombinant saccharomyces cerevisiaes, construction methods of recombinant saccharomyces cerevisiaes and applications of recombinant saccharomyces cerevisiaes in production of hydroxy fatty acids

A technology for producing hydroxy fatty acid and Saccharomyces cerevisiae, which is applied in the biological field

Pending Publication Date: 2020-05-08
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the heterologous synthesis of ω(ω-1)-hydroxy fatty acids with specific structures (C16-C18) in Saccharomyces cerevisiae has not been reported using synthetic biology methods

Method used

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  • Recombinant saccharomyces cerevisiaes, construction methods of recombinant saccharomyces cerevisiaes and applications of recombinant saccharomyces cerevisiaes in production of hydroxy fatty acids
  • Recombinant saccharomyces cerevisiaes, construction methods of recombinant saccharomyces cerevisiaes and applications of recombinant saccharomyces cerevisiaes in production of hydroxy fatty acids
  • Recombinant saccharomyces cerevisiaes, construction methods of recombinant saccharomyces cerevisiaes and applications of recombinant saccharomyces cerevisiaes in production of hydroxy fatty acids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 A kind of construction method of recombinant Saccharomyces cerevisiae (1) producing hydroxy fatty acid

[0037] module construction

[0038] According to the DNA sequences of the acyl-CoA oxidase encoding gene POX1, the acyl-CoA activating enzyme encoding gene FAA1 and the acyl-CoA activating enzyme encoding gene FAA4 provided on NCBI, design homology arms POX1A-up, POX1B-down, FAA1A-up , FAA1B-down, FAA4A-up, FAA4B-down. The templates were all from the genome of Saccharomyces cerevisiae BY4741; the selection marker gene his3 was from the plasmid Pxp320 (purchased from Addgene, Inc. www.addgene.org ).

[0039] Using the Saccharomyces cerevisiae BY4741 genome as a template,

[0040] With POX1A-F (SEQ ID NO.12),

[0041] HIS3-POX1A-R (SEQ ID NO.13) as primer

[0042] Amplified homology arm POX1A-up (SEQ ID NO.14)

[0043] With HIS3-POX1B-F (SEQ ID NO.15),

[0044] POX1B-R (SEQ ID NO.16) is a primer,

[0045] Amplified homology arm POX1B-down (SEQ ID ...

Embodiment 2

[0083] Embodiment 2, a kind of construction method of the recombinant Saccharomyces cerevisiae that produces hydroxy fatty acid (two)

[0084] Using the Saccharomyces cerevisiae BY4741 genome as a template, HOA-F (SEQ ID NO.38), PGK1p-HOA-R (SEQ ID NO.39) and HIS3-HOB-F (SEQ ID NO.40), HOB-R (SEQ ID NO.41) as primers to amplify the homology arms HOup (SEQ ID NO.42) and HOdown (SEQ ID NO.43); -R (SEQ ID NO.45) and CYP52M1-ADH1t-F (SEQ ID NO.46), TDH3p-ADH1t-R (SEQ ID NO.47) are primers to amplify the promoter P PGK1 (SEQ ID NO.7) and terminator T ADH1t (SEQ ID NO.8); using PGK1p-CYP52M1-F (SEQ ID NO.48) and ADH1t-CYP52M1-R (SEQ ID NO.49) as primers to amplify the cytochrome P450 oxidase gene CYP52M1 (SEQ ID NO .4); respectively with ADH1t-TDH3p-F (SEQ ID NO.50), SbCPR-TDH3p-R (SEQ ID NO.51) and SbCPR-TDH2t-F (SEQ ID NO.52), HIS3-TDH2t-R (SEQ ID NO.53) is a primer to amplify the promoter P TDH3 (SEQ ID NO.9) and terminator T TDH2t (SEQ ID NO.10); using TDH3p-SbCPR-F (SEQ ID...

Embodiment 3

[0086] Example 3, the construction method of recombinant Saccharomyces cerevisiae (three) producing hydroxy fatty acid

[0087] Using the Saccharomyces cerevisiae BY4741 genome as a template, HOA-F (SEQ ID NO.38), PGK1p-HOA-R (SEQ ID NO.39) and HIS3-HOB-F (SEQ ID NO.40), HOB-R (SEQ ID NO.41) as primers to amplify the homology arms HOup (SEQ ID NO.42) and HOdown (SEQ ID NO.43); -R (SEQ ID NO.45) and CYP52M1-ADH1t-F (SEQ ID NO.46), TDH3p-ADH1t-R (SEQ ID NO.47) are primers to amplify the promoter P PGK1 (SEQ ID NO.7) and terminator T ADH1t (SEQ ID NO.8); using PGK1p-CYP52M1-F (SEQ ID NO.48) and ADH1t-CYP52M1-R (SEQ ID NO.49) as primers to amplify the cytochrome P450 oxidase gene CYP52M1 (SEQ ID NO .4); respectively with ADH1t-TDH3p-F (SEQ ID NO.50), AtCPR1-TDH3p-R (SEQ ID NO.58) and AtCPR1-TDH2t-F (SEQ ID NO.59), HIS3-TDH2t-R (SEQID NO.53) is a primer to amplify the promoter P TDH3 (SEQ ID NO.9) and terminator T TDH2t (SEQ ID NO.10); with TDH3p-AtCPR1-F (SEQ ID NO.60) and TD...

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Abstract

The invention discloses recombinant saccharomyces cerevisiaes, construction methods of the recombinant saccharomyces cerevisiaes and applications of the recombinant saccharomyces cerevisiaes in production of hydroxy fatty acids. Through a homologous recombination method, the recombinant fungi (1) are obtained by knocking out key gene acyl coenzyme A oxidase POX1 in a beta-oxidation pathway relatedto fatty acid catabolism in saccharomyces cerevisiaes and knocking out acyl coenzyme A activating enzymes FAA1 and FAA4, recombinant fungi (2) are obtained by introducing cytochrome P450 oxidase CYP52M1 and cytochrome P450 reductase SbCPR into the recombinant fungi (1), or the recombinant fungi (3) are obtained by introducing cytochrome P450 oxidase CYP52M1 and cytochrome P450 reductase AtCPR1 into the recombinant fungi (1), or the recombinant fungi (4) are obtained by introducing cytochrome P450 oxidase and cytochrome P450 reductase fusion protein gene CYP52M1-AtCPR1 into the recombinant fungi (1).

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of recombinant Saccharomyces cerevisiae and its construction method and production of hydroxy fatty acid. Background technique [0002] Hydroxy fatty acids are a class of small molecular compounds with carboxyl and hydroxyl groups, which have better reactivity, stability and viscosity than fatty acids. Due to their unique properties, long-chain hydroxy fatty acids are widely used in various fields, such as food, chemical, and cosmetic industries, and as intermediates in the synthesis of ceramides, polyesters, and cyclic lactones. Pharmaceutical properties of hydroxy fatty acids, including antibiotic, anti-inflammatory and antidiabetic effects were also revealed. In particular, the different positions of hydroxyl groups in hydroxy fatty acid molecules play a crucial role in influencing physiological properties and chemical industry. Hydroxylation close to the carboxy...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/90C12N15/53C12N15/54C12N15/62C12N1/19C12P7/42C12R1/865
CPCC12N15/81C12N15/905C12N9/001C12N9/1029C12N9/0042C12N9/0071C12P7/42C12Y106/02004C12Y103/03006C12Y114/14C07K2319/00
Inventor 卢文玉李冰刘静静张传波
Owner TIANJIN UNIV
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