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Recombinant yeast strain, and construction method and application thereof

A technology of yeast strain and construction method, applied in the field of genetic engineering, to achieve the effect of increasing synthetic yield

Active Publication Date: 2016-03-23
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there is still a certain gap compared with the highest yield (50.6mg / gDCW) of lycopene synthesized by recombinant Escherichia coli previously reported.

Method used

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  • Recombinant yeast strain, and construction method and application thereof
  • Recombinant yeast strain, and construction method and application thereof
  • Recombinant yeast strain, and construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0119] Example 1: Construction of gene knockout strains

[0120]Using Saccharomyces cerevisiae CEN.PK2-1D as the starting strain, a four-gene knockout strain CEN.PK2-1CΔgal1,Δgal7,Δgal10::DR,Δypl062w::kanMX was constructed. The specific process is as follows:

[0121] First construct Δgal1, Δgal7, Δgal10::DR-KlURA3-DR knockout cassettes, that is, knockout cassette fragment 1, and use plasmid pWJ1042 as a template to design upstream and downstream primers for PCR amplification with 40bp homology arms and DR - KlURA3-DR nutritional label knockout box fragment, using the yeast's own homologous recombination mechanism to integrate the fragment into the yeast genome through lithium acetate yeast transformation, and use SD-URA solid plate after transformation (synthetic yeast nitrogen source YNB6. 7g / L of glucose, 20g / L of glucose, 2g / L of mixed amino acid powder lacking uracil, 2% agar powder) to carry out screening, the transformants obtained carry out PCR verification by extract...

Embodiment 2

[0123] Embodiment 2: Construction of gene fragment 1

[0124] Amplify the CYC1 terminator, BtcrtI, GAL10 promoter, GAL1 promoter, PacrtB, PGK1 terminator and splice them sequentially by OE-PCR method to obtain a fragment T containing HindIII and XhoI restriction sites at both ends CYC1 -crtI-P GAL10 -P GAL1 -crtB-T PGK1 ;

[0125] At the same time, the homologous 631bp sequence upstream of the yeast trp1 site and the 733bp homologous sequence downstream of the yeast trp1 site were amplified and spliced ​​sequentially by OE-PCR to obtain SacI and ApaI restriction sites at both ends, and in yeast trp1 The fragment containing the HindIII and XhoI restriction sites between the upstream and downstream homologous sequences of the site is then connected into the vector pRS405 through the SacI and ApaI restriction sites (see SEQ ID NO: 6 for the complete gene sequence, and see Figure 9 ), to obtain the TRP1 integration plasmid pRS405-TRP, the above obtained fragment T CYC1 -crtI...

Embodiment 3

[0128] Embodiment 3: the construction of gene fragment 2

[0129] The ACT1 terminator, the truncated HMG-CoA reductase gene tHMGR1, the GAL10 promoter, the GAL1 promoter, the TMcrtE, and the GPM1 terminator were sequentially spliced ​​together by OE-PCR, and both ends contained BamHI and XhoI restriction sites Fragment T ACT1 -tHMGR1-P GAL10 -P GAL1 -crtE-T GPM1 At the same time, the upstream homologous 561bp sequence of the yeast leu2 site, the LEU2 marker, the TDH2 terminator, and the downstream homologous 584bp sequence of the yeast leu2 site were spliced ​​sequentially by OE-PCR method to obtain SacI and ApaI restriction sites at both ends point, and the fragment containing the BamHI and XhoI restriction sites between the TDH2 terminator and the downstream homologous sequence of the yeast leu2 site, was connected into the vector pRS405 through the SacI and ApaI restriction sites to obtain the LEU2 integration plasmid pRS405-LEU. will result in the above fragment T ACT...

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Abstract

The invention relates to the technical field of gene engineering, and discloses a recombinant yeast strain, and a construction method and an application thereof. The recombinant yeast strain is obtained through the following steps: knocking out gal1, gal7, gal10 and ypl062w genes, integrating three gene fragments a genome through yeast homologous recombination, further knocking out an rox1 gene, and further integrating two gene fragments to the yeast genome. The gene knockout yeast strain constructed in the invention provides optimized host cells for production of lycopene; and combined functional genes crtE, crtB and crtI having specific sources and used for synthesizing the lycopene and specific yeast endogenous genes are selected and are integrated to the gene knockout strain genome through modular designing, so the brand new recombinant strain for highly yielding lycopene is obtained.

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] Worldwide, with the improvement of the economic level and the demand for health, more and more attention has been paid to the safety, nutrition and functionality of food. Therefore, functional nutritional chemicals have become a development trend, representing the development of contemporary food. The new trend has a broad market prospect. Lycopene is a fat-soluble natural food coloring, which belongs to carotenoids in chemical structure and has strong antioxidant capacity. It has become a hot spot in the research of functional food ingredients in the world in recent years. [0003] The preparation of lycopene mainly depends on plant extraction, chemical synthesis and microbial synthesis. The first two methods have their own shortcomings. Microbial sy...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P5/02C12R1/865C12R1/645
CPCC12N9/001C12N9/1085C12P5/026C12Y103/05005C12Y205/01029C12Y205/01032
Inventor 肖文海陈艳李霞元英进
Owner TIANJIN UNIV
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