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Recombinant yeast strain as well as construction method and application thereof

A yeast strain and yeast technology, applied in the field of genetic engineering, can solve problems such as gaps, and achieve the effect of improving synthetic yield

Active Publication Date: 2015-11-25
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still a big 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 as well as construction method and application thereof
  • Recombinant yeast strain as well as construction method and application thereof
  • Recombinant yeast strain as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1: Construction of gene knockout strains

[0100] Using Saccharomyces cerevisiae CEN.PK2-1C as the starting strain, a four-gene knockout strain CEN.PK2-1C△gal1,△gal7,△gal10::DR,△ypl062w::kanMX was constructed. The specific process is as follows:

[0101] First construct △gal1, △gal7, △gal10::DR-KlURA3-DR knockout cassettes, that is, knockout cassette fragment 1, and use the plasmid pWJ1042 as a template to design upstream and downstream primers for PCR amplification with 40 bp homology of the gene upstream and downstream Arm and DR-KlURA3-DR nutritional tag knockout box fragment, using the homologous recombination mechanism of the yeast itself, the fragment was transformed into the yeast genome through lithium acetate yeast transformation, and after transformation, SD-URA solid plate (synthetic yeast nitrogen Source YNB6.7g / L, glucose 20g / L, mixed amino acid powder 2g / L lacking uracil, 2% agar powder) is screened, and the obtained transformants are subjected to...

Embodiment 2

[0104] Embodiment 2: Construction of gene knockout strain

[0105] Using Saccharomyces cerevisiae CEN.PK2-1C as the starting strain, a double gene knockout strain CEN.PK2-1C△gal80::DR,△ypl062w::kanMX was constructed. The specific process is as follows:

[0106] First construct the △gal80::DR-KlURA3-DR knockout cassette, that is, the knockout cassette fragment 2, and use the plasmid pWJ1042 as a template to design the upper and lower primers for PCR amplification with the upper and lower 40bp homology arms of the gene and the DR-KlURA3- The fragment of the knockout cassette of the DR nutrition label is integrated into the yeast genome by using the homologous recombination mechanism of yeast itself through yeast transformation with lithium acetate method. After transformation, SD-URA solid plate (synthetic yeast nitrogen source YNB6.7g / L , glucose 20g / L, single uracil-deficient mixed amino acid powder 2g / L, 2% agar powder) for screening, the obtained transformants were purified...

Embodiment 3

[0108] Embodiment 3: Construction of gene fragment 1

[0109] 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 ;

[0110] 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: 8 for the complete gene sequence, and see Figure 8 ), to obtain the TRP1 integration plasmid pRS405-TRP, the above obtained fragment T CYC1 -crtI...

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Abstract

The invention relates to the technical field of genetic engineering, and discloses a recombinant yeast strain as well as a construction method and an application thereof. The recombinant yeast strain has the knockout gene gal1, gal7, gal10 or gal80, and the knockout gene ypl062w, and contains 2-4 gene segments integrated to the genome through the yeast homologous recombination. According to the invention, the yeast strain with the knockout genes is established, an optimized host cell is provided for producing lycopene, and functional genes crtE, crtB and crt I with different sources for synthetizing lycopene, and the specific yeast endogenous genes are selected, and integrated to the genome of the yeast strain with the knockout gene through the modular design, and thus a brand-new recombinant strain with high yield of 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. A new trend with broad market prospects. 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 synt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P5/02
Inventor 肖文海陈艳李霞元英进
Owner TIANJIN UNIV
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