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

A technology of Saccharomyces cerevisiae strains and strains, applied in the field of genetic engineering, to achieve the effect of increasing synthetic yield

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

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Problems solved by technology

Chinese patent CN105087406A discloses a recombinant yeast strain and its construction method and application, which is achieved by knocking out the yeast strain gal1 , gal7 , gal10 or gal80 genes, and ypl062w Genetic construction of gene knockout yeast strains, and then selection of functional genes for lycopene synthesis from different sources crtE, crtB and crtI , and specific yeast endogenous genes, etc., were integrated into the genome of the gene-knockout yeast strain through modular design, and a brand-new recombinant strain with high lycopene production was obtained, and the lycopene production reached 30-45mg / gDCW; however , which is still far from the highest yield (50.6 mg / g DCW) of lycopene synthesized by recombinant Escherichia coli previously reported.

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

Examples

Experimental program
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Embodiment 1

[0092] Example 1: Construction of gene knockout strains

[0093] Using Saccharomyces cerevisiae CEN.PK2-1D as the starting strain, construct the four-gene knockout strain CEN.PK2-1C △gal1,△ gal7,△gal10::DR , △ypl062w::kanMX . The specific process is as follows:

[0094] build first Δgal1, Δgal7, Δgal10::DR-Kl URA3-DR Knockout box, i.e. knockout box fragment 1, with plasmid pWJ1042 as a template, design upstream and downstream primers PCR amplification band gene upper and lower stream 40bp homology arms and DR-K1 URA3-DR nutrition label knockout box fragment, utilize The yeast's own homologous recombination mechanism integrates the fragment into the yeast genome through lithium acetate yeast transformation. After transformation, SD-URA solid plate (synthetic yeast nitrogen source YNB 6.7g / L, glucose 20g / L, single-deficient uracil mixed amino acid powder 2g / L, 2% agar powder) for screening, the obtained transformants were purified and cultured and then the yeast genome ...

Embodiment 2

[0096] Embodiment 2: Construction of gene fragment 1

[0097] Amplify the CYC1 terminator, Bt crtI , GAL10 promoter, GAL1 promoter, Pa crtB , PGK1 terminator and sequentially spliced ​​together by OE-PCR method to obtain two ends containing Hind III and xho Fragment T of I Restriction Site CYC1 -crtI-P GAL10 -P GAL1 -crtB-T PGK1 ;

[0098] At the same time, the homologous 631bp sequence upstream of the yeast trp1 site and the 733bp sequence homologous downstream of the yeast trp1 site were amplified and spliced ​​sequentially by OE-PCR to obtain Sac I and Apa I enzyme cutting site, and contains between the yeast trp1 site and downstream homologous sequences Hind III and xho Fragmentation of the I restriction site, followed by Sac I and Apa The I restriction site is connected into the carrier pRS405 to obtain the TRP1 integration plasmid pRS405-TRP, and the fragment TRP obtained above is CYC1 -crtI-P GAL10 -P GAL1 -crtB-T PGK1 with pRS405-TRP plasmid b...

Embodiment 3

[0101] Embodiment 3: the construction of gene fragment 2

[0102] ACT1 terminator, truncated HMG-CoA reductase gene tHMGR1 , GAL10 promoter, GAL1 promoter, TMcrtE, GPM1 terminators were spliced ​​sequentially by OE-PCR method to obtain two ends containing Bam H I and xho Fragment T of I Restriction Site 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 to obtain the two ends containing Sac I and Apa I restriction site, and contains between TDH2 terminator, downstream homologous sequence of yeast leu2 site Bam H I and xho Fragments of the I restriction site, passed through Sac I and Apa The I restriction site was connected into the vector pRS405 to obtain the LEU2 integration plasmid pRS405-LEU. will result in the above fragment T ACT1 -tHMGR...

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Abstract

The invention relates to the technical field of gene engineering, and discloses a recombinant yeast strain, a construction method thereof and the application thereof. In the recombinant yeast strain, gal1, gal7, gal10 and ypl062w genes are knocked out, and the recombinant yeast strain comprises 6 gene segments which are integrated to the genome through yeast homologous recombination. On the above basis, the recombinant yeast strain is further integrated onto one gene segment of a yeast genome, so that a better recombinant yeast is obtained. According to the technical scheme of the invention, the yeast strain is constructed based on gene knock-out, and an optimized host cell for producing lycopene is provided. Meanwhile, functional genes crtE crtB and crtI of a specific source combination and used for the synthesis of lycopene, specific yeast endogenous genes and specific exogenous genes are selected. The above selected genes are integrated to the gene knock-out genome of the yeast strain, and then a brand-new recombinant strain capable of producing high-yield 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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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81C12P1/02C12R1/865C12R1/645
CPCC07K14/39C07K14/395C12N9/16C12N9/2402C12N15/81C12P1/02C12Y302/01023
Inventor 李霞陈艳肖文海王颖姚明东刘宏元英进
Owner TIANJIN UNIV
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