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Recombinant yeast strain, and 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: 2015-10-07
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 between the production of β-carotene synthesized by recombinant Saccharomyces cerevisiae and recombinant E. coli reported so far

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

[0129] Embodiment 1: Construction of four knockout strains

[0130] 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:

[0131] First construct △gal1, △gal7, △gal10::DR-Kl URA3-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 the same 40 bp upstream and downstream of the gene. The knockout box fragment of the source arm and the DR-K1 URA3-DR nutritional label was 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 YNB 6.7g / L, glucose 20g / L, mixed amino acid powder 2g / L lacking uracil, 2% agar powder) were screened, and the obtained transforma...

Embodiment 2

[0133] Embodiment 2: Construction of gene fragment 1

[0134] Amplify the CYC1 terminator, Bt crtI, GAL10 promoter, GAL1 promoter, Pa crtB, PGK1 terminator and splice them sequentially by OE-PCR to obtain a fragment T containing HindIII and XhoI restriction sites at both ends CYC1 -crtI-P GAL10 -P GAL1 -crtB-T PGK1 ;

[0135]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: 15 for the complete gene sequence, and see Figure 12 ), to obtain the TRP1 integration plasmid pRS405-TRP, the above obtained fragment T CYC1 -crtI-P ...

Embodiment 3

[0138] Embodiment 3: the construction of gene fragment 2

[0139] Design upstream and downstream primers from the SyBE_Sc0014C012 genome to PCR amplify the kanMX resistance tag upstream 123bp homologous sequence and downstream 538bp homologous sequence respectively, and PCR amplify the DR-K1 URA3-DR nutritional label from the plasmid pWJ1042; Upstream homologous sequence, DR-K1 URA3-DR nutritional label, T constructed in Example 2 CYC1 -crtI-P GAL10 -P GAL1 -crtB-T PGK1 The fragment and the downstream homologous sequence of the kanMX resistance tag were spliced ​​sequentially by OE-PCR method to obtain the integrated fragment of gene segment 2 containing PmeI restriction sites at both ends, that is, the upstream homologous sequence of the kanMX resistance tag-DR-Kl URA3 -DR-T CYC1 -crtI-P GAL10 -P GAL1 -crtB-T PGK1 -kanMX resistance tag downstream homologous sequence, and connected with the blunt end vector pJET1.2 to obtain the kanMX integration plasmid pkanMX, denoted...

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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 application thereof. In the recombinant yeast strain, gal1, gal7, gal10 and ypl062w genes are knocked out, and the recombinant yeast strain comprises 9 gene segments which are integrated to the genome by yeast homologous recombination. The construction method comprises the following steps: constructing a four-knock-out yeast strain to provide an optimized host cell for producing beta-carotin, selecting lycopene cyclases crtY from different sources, and carrying out modular design to integrate functional genes crtE crtB and crtI of the specific-source synthetic lycopene, specific yeast endogenous genes and the like to the four-knock-out yeast strain genome, thereby obtaining a brand-new recombinant strain capable of producing beta-carotin at high yield.

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. β-carotene is an orange-yellow fat-soluble natural food coloring with strong antioxidant capacity, and has become a hot spot in the research of functional food ingredients in the world in recent years. [0003] The preparation of β-carotene mainly relies on plant extraction, chemical synthesis and microbial synthesis. The first two methods have their own shortcomings. Microbial synthesis is considered to be the most...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12P23/00C12R1/865C12R1/645
Inventor 肖文海陈艳李霞元英进
Owner TIANJIN UNIV
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