Method for rapid optimization of yeast cell plants

A yeast cell and yeast technology, applied in the field of rapid optimization of yeast cell factories, can solve problems such as low purity of beta-carotene, and achieve the effects of low cost, high efficiency and improved efficiency

Inactive Publication Date: 2018-01-30
TIANJIN UNIV
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Problems solved by technology

At the same time, Yu Hongwei's research group at Zhejiang University applied an inducible expression system to achieve a total carotenoid production of 11mg / gDCW (7.41mg / gDCWβ-carotene) in Saccharomyces cerevisiae; Optimizing the means, and improving the output of total carotene to 1156mg / L (20.79mg / gDCW) through fermentation optimization, but wherein the purity of β-carotene is low, only accounting for 30% of the total output

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  • Method for rapid optimization of yeast cell plants
  • Method for rapid optimization of yeast cell plants

Examples

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Embodiment 1

[0031] Example 1: Construction of recombinant Saccharomyces cerevisiae strains producing β-carotene

[0032] Taking the yeast strain carrying the synV chromosome as the starting strain, four genes for the synthesis of β-carotene from different sources were obtained from GeneBank: GGPP synthase gene crtE, phytoene synthase gene crtB, The phytoene dehydrogenase gene crtI and the lycopene cyclase gene crtY, meanwhile, in order to integrate the four genes into the chromosome of Saccharomyces cerevisiae for stable expression, a yeast endogenous assembly strategy was adopted. About 40 bp of homology arms were designed between the obtained above gene fragments, and at the same time, the genes on the left and right sides were designed to be homologous to the CAN site of Saccharomyces cerevisiae synV chromosome, with a length of about 40 bp. The above-mentioned genes were all codon-optimized by Saccharomyces cerevisiae and properly avoided common restriction enzyme sites, and were arti...

Embodiment 2

[0033] Example 2: Optimization of recombinant Saccharomyces cerevisiae strain yYW245 producing β-carotene

[0034] 1. Construction of diploid Saccharomyces cerevisiae strain

[0035] Saccharomyces cerevisiae yYW0245(MAT a) was hybridized with artificial Saccharomyces cerevisiae yYW0233(MATα) carrying chromosome number synIII, wherein the 3' end of each complete functional element on chromosome number synIII was inserted into the nucleotide sequence shown in SEQ ID NO:1 (Using de novo synthesis to insert the nucleotide sequence shown in SEQ ID NO:1 into the 3' end of the original chromosome functional element on a large scale), the specific steps for the construction of the diploid Saccharomyces cerevisiae strain are as follows:

[0036] After the two kinds of yeast plates were streaked and activated, they were inoculated into 5ml YPD (20g / L peptone, 10g / L yeast powder, 22g / L glucose monohydrate, 20g / L agar powder, pH 6.1) liquid medium at the same time, at 30°C After culturin...

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Abstract

The invention relates to the technical field of biology, in particular to a method for rapid optimization of yeast cell plants. The feature that diploids can be formed among haploid brewing yeasts through mating is utilized, the specific recombination technology of a Cre-LoxP system is improved, and genome rearrangement efficiency is greatly improved, target strains are simply and rapidly optimized and high-producing strains are screen out on the basis of a yeast strain library rearranged by a large number of genomes. Compared with a certain gene optimization technology in the prior art, the method is popular, low in cost and efficient.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for rapidly optimizing a yeast cell factory. Background technique [0002] With economic growth, social progress and further improvement of medical level, people pay more attention to their health, especially the market demand for well-made, safe and efficient, high-quality and cheap functional nutritional chemicals and cosmetics has further expanded. In the context of global resource shortage and environmental pollution, the traditional chemical industry is increasingly unable to meet the requirements of sustainable development due to problems such as high energy consumption and serious pollutant emissions. [0003] With the popularization of DNA sequencing technology, the whole genome information of more and more species has been revealed. In addition, the development of related omics technologies such as transcriptome, proteome, and metabolome has enabled scientists to un...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/04C12N15/65C12N15/90C12R1/865
Inventor 元英进吴毅王娟靳瑾
Owner TIANJIN UNIV
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