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A kind of recombinant yeast strain and its construction method and application

A technology of yeast and strains, applied in the field of genetic engineering, can solve the problems of increasing costs and not being able to improve the economic benefits of β-carotene, and achieve the effect of high yield

Active Publication Date: 2019-04-16
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the recombinant yeast strain obtained in the patent CN 104962488A uses a Gal-inducible promoter and a gene module integrated with the inducible promoter. If the recombinant Saccharomyces cerevisiae produces β-carotene, it needs to be induced by adding galactose, while Galactose is a relatively expensive raw material, which will increase the cost in actual production and cannot improve the economic benefits of β-carotene, and there is no related report on the recombinant Saccharomyces cerevisiae with a constitutive promoter

Method used

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  • A kind of recombinant yeast strain and its construction method and application
  • A kind of recombinant yeast strain and its construction method and application
  • A kind of recombinant yeast strain and its construction method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1: Construction of gene fragment 3 and gene fragment 5 (construction of upstream and downstream homology arms of Delta site)

[0072] The 248bp homologous sequence Delta1 upstream of the Delta site, the URA3 tag DRURA3DR with a frameable frame (5-FOA can be used for screening to knock out the strain of the screening marker) and the promoter TEF1p were spliced ​​together by OE-PCR method and spliced ​​at both ends Add the Not I restriction site and connect it into the vector pEasyBlunt to obtain the upstream homology arm vector of the Delta site (gene fragment 3 can be obtained after restriction);

[0073] The terminator TDH2t and the 239bp homologous sequence Delta2 downstream of the Delta site were spliced ​​together by the OE-PCR method, and Not I restriction sites were added at both ends, and connected into the vector pEasyBlunt to obtain the downstream homology arm vector of the Delta site ( The gene fragment 5) can be obtained after digestion.

[0074] ...

Embodiment 2

[0081] Embodiment 2: Construction of gene fragment 4

[0082] Step 1.1, splicing the promoter TEF1p, Xd CrtE, and terminator PDX1t sequentially by OE-PCR method to obtain the fragment P TEF1p -Xd CrtE-T PDX1t ,spare;

[0083] The terminator PDX1t homologous sequence, promoter TDH3p, Xd CrtI, terminator MPE1t, and promoter FBA1p homologous sequences were spliced ​​sequentially by OE-PCR to obtain the fragment terminator PDX1t homologous sequence-P TDH3p -Xd CrtI-T MPE1t -Promoter FBA1p homologous sequence, spare;

[0084] The promoter FBA1p, Xd CrtYB, and terminator TDH2t were sequentially spliced ​​together by OE-PCR to obtain the fragment P FBA1p -Xd CrtYB-T TDH2t ,spare;

[0085] Step 1.2, by PCR in fragment P TEF1p -Xd CrtE-T PDX1t Add 40 bp of the homologous sequence upstream of the EcoRI restriction site in the vector pRS416 and the EcoR Ⅰ restriction site before the promoter of the vector pRS416, and obtain the upstream homologous sequence of the EcoR Ⅰ restricti...

Embodiment 3

[0088] Example 3: Integration of gene segments 3-5 to construct a transitional recombinant Saccharomyces cerevisiae strain

[0089] The gene fragment 3-5 was transferred into BY4741 yeast by the lithium acetate method, and the homologous sequence in the gene fragment 3-5 was connected into gene fragment 1, namely Delta1-URA3 tag-P TEF1p -Xd CrtE-T PDX1t -P TDH3p -Xd CrtI-T MPE1t -P FBA1p -Xd CrtYB-T TDH2t -Delta2, and then integrated into the genome through recombination between the upstream and downstream homologous sequences of the Delta site and the Delta site on the yeast genome (see the schematic diagram figure 1 ), to obtain a transitional recombinant yeast strain;

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Abstract

The invention relates to the technical field of gene engineering, and discloses a recombinant yeast strain, and a building method and application thereof. The recombinant yeast strain includes two specific gene segments recombined and integrated onto a genome in a homologous way through yeast. By selecting the specific group for forming a promoter combination and matching phaffia rhodozyma sourced phytoene synthetase and lycopene cyclase dual-function enzyme gene CrtYB, GGPP synthase CrtE, phytoene desaturase CrtI and specific yeast endogenous gene and the like, the substances are integrated onto the yeast strain genome through modular design; the yp1062w gene is knocked away through homologous recombination; one strain of fire-new recombinant yeast without an inducing agent is obtained; the high yield of the beta-renieratene is ensured.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, and more specifically relates to a recombinant yeast strain and its construction method and application. Background technique [0002] Carotenoids are a class of commercial pigments with important physiological functions. In the human body, β-carotene (β-carotene) is the precursor of vitamin A, which has the potential functions of anti-oxidation, cancer prevention and immune enhancement. They are also commonly used as coloring agents in food and feed industries. At present, chemical synthesis of β-carotene still occupies most of the market, but due to the hazards of chemical reagents, people are looking for other alternative methods; large-scale cultivation of algae and fungi that naturally produce carotenoids is another alternative to carotenoid production. It is a main method, but it also has the problems of high cost and serious pollution. Therefore, the synthesis of carotenoids ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/87C12P23/00
CPCC07K14/39C12N9/0006C12N9/001C12N9/1085C12N9/90C12P23/00C12Y101/01034C12Y103/05005C12Y205/0101C12Y205/01029C12Y205/01032C12Y505/01
Inventor 肖文海王瑞钊潘才惠贾斌元英进
Owner TIANJIN UNIV