Method for rapidly improving tolerance of yeast

A tolerance, yeast technology, applied in the biological field, can solve the problem of small strain screening library, and achieve the effect of improving the evolution speed and efficiency, improving the possibility, and eliminating the operation of in vitro molecular construction

Inactive Publication Date: 2018-01-05
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the current strain transformation method also has the disadvantage of a small strain screening library.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: Improve the high temperature resistance performance of Saccharomyces cerevisiae Y12

[0024] 1. Obtaining Saccharomyces cerevisiae diploid:

[0025] The Y12 Saccharomyces cerevisiae of the mating type a and the haploid Saccharomyces cerevisiae strain yYW0139 to be mated with the nucleotide sequence shown in SEQ ID NO: 1 inserted at the 3' end of each complete functional element of the mating type α were simultaneously inoculated into 5ml YPD The mating was carried out in liquid culture, and the diploid Saccharomyces cerevisiae strains were picked out by a microscope and verified by a microscope.

[0026] 2. Yeast transformation with Cre enzyme expression system plasmid:

[0027] The plasmid containing the Cre recombinase expression cassette (with the ura nutritional tag) was transformed into the diploid Saccharomyces cerevisiae strain in the previous step using lithium acetate chemical transformation method.

[0028] 3. Expression of Cre recombinase and ...

Embodiment 2

[0032] Embodiment 2: Improve the drug resistance (rapamycin) performance of Saccharomyces cerevisiae CBS5829

[0033]1. Obtaining Saccharomyces cerevisiae diploid:

[0034] CBS5829 Saccharomyces cerevisiae of mating type a and haploid Saccharomyces cerevisiae strain yYW0139 to be mated at the 3' end of each non-essential gene inserted into the nucleotide sequence shown in SEQ ID NO: 1 were simultaneously inoculated into The mating was carried out in 5ml YPD liquid culture, and the diploid Saccharomyces cerevisiae strain was picked out by a microscope sporulation instrument and verified.

[0035] 2. Yeast transformation with Cre enzyme expression system plasmid:

[0036] The plasmid containing the Cre recombinase expression cassette (with the ura nutritional tag) was transformed into the diploid Saccharomyces cerevisiae strain in the previous step using lithium acetate chemical transformation method.

[0037] 3. Expression of Cre recombinase and on / off of site-specific recomb...

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Abstract

The invention relates to the technical field of biology, and particularly discloses to a method for rapidly improving the tolerance of yeast. According to the method, by utilizing the characteristic that haploid brewer's yeast can form diploids by mating and an improved specificity recombination technology of a Cre-LoxP system, the construction operation of in-vitro molecules is not involved, theevolution speed and efficiency are greatly increased, the specificity recombination of loci of the whole genome level is achieved, a large number of yeast strain libraries the genomes of which are rearranged are obtained, and the possibility of the dominance phenotype from high-throughput screening to evolution is significantly increased.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a method for rapidly improving yeast tolerance. Background technique [0002] Strain modification technology is a technology that uses the principles of genetics and biochemistry to carry out molecular modification of target strains and screen for corresponding conditions, so as to provide excellent strains for production and promote production development. Strain transformation technology currently mainly includes gene mutation (such as mutagenesis, etc.), gene recombination (such as hybridization, protoplast fusion, genetic engineering, etc.), direct gene evolution (such as error-prone PCR, DNA shuffling, exon shuffling, etc.), etc. method. [0003] However, there are many shortcomings in the current strain transformation methods. The gene mutation method has a certain degree of blindness and randomness, and the target of the operation is not clear; most of the gen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/65C12N15/90C12R1/865
Inventor 元英进吴毅张皓然李云祥徐晖
Owner TIANJIN UNIV
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