Gene element and application of same to variation of chromosome number, copy number and structure of Saccharomyces cerevisiae

A genetic element, technology of Saccharomyces cerevisiae, applied in the field of synthetic biology, can solve the problem of high lethality

Active Publication Date: 2018-11-30
TIANJIN UNIV
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  • Description
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Problems solved by technology

At present, using this system to carry out genome rearrangement in diploid synthetic Saccharomyces cerevisiae has achieved multiple rounds of increase in β-carotene production, but due to the high lethality of haploid genome rearrangement, it has not yet been used in a single Multiple rounds of increased production of secondary metabolites achieved in ploidy synthetic Saccharomyces cerevisiae

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  • Gene element and application of same to variation of chromosome number, copy number and structure of Saccharomyces cerevisiae
  • Gene element and application of same to variation of chromosome number, copy number and structure of Saccharomyces cerevisiae
  • Gene element and application of same to variation of chromosome number, copy number and structure of Saccharomyces cerevisiae

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Embodiment 1

[0053] Embodiment 1 Haploid Saccharomyces cerevisiae phenotype continuous evolution method

[0054] The invention provides a research method for accelerating the phenotypic evolution of strains by using synthetic Saccharomyces cerevisiae circular chromosomes, using PDV, a downstream metabolite of aromatic amino acids, as a marker to generate a large number of phenotypic variant yeast strains by inducing circular chromosome rearrangement.

[0055] 1. design primers PCR amplify the open reading frame (ORF) of vioA, vioB and vioE from the biological building block (BioBrick) component library, agarose gel reclaims DNA fragment.

[0056] 2. Design primers to amplify the promoter and terminator from Saccharomyces cerevisiae BY4741 genomic DNA, wherein there is an overlapping region of 40 base pairs (bp) between the promoter, open reading frame and terminator. DNA fragments were recovered from agarose gel.

[0057] 3. The pRS416 plasmid was digested with EcoRI restriction endonucle...

Embodiment 2

[0079] Example 2 Saccharomyces cerevisiae violacein precursor metabolism strain continuous improvement

[0080] Circular synthetic yeast chromosomal rearrangements can achieve sustained improvements in the phenotype of yeast strains. We performed 5 rounds of rearrangement experiments on the strain (yWJS067), expecting to obtain a strain with improved PDV production. For the finally selected strains, the percentages of dark colonies were 7.79%, 3.76%, 4.09%, 3.88%, 3.84% and 1.07%, respectively. For each round of rearrangement experiments, the strain with the darkest visible color and similar growth fitness compared to the parental strain was selected for quantification of PDV productivity by HPLC. After six rounds of rearrangement experiments, we obtained a strain with about seven-fold increase in PDV production, and the strain had a good growth phenotype.

[0081] Table 4 figure 2 aRaw data

[0082]

[0083] table 5 figure 2 bRaw data

[0084]

[0085] Ring synthe...

Embodiment 3

[0086] Example 3 Saccharomyces cerevisiae chromosome number continuous variation

[0087]In order to reveal the genetic variation generated in ring_synV in depth, the applicant conducted whole genome sequencing (WGS) analysis on some induced rearrangement strains (SCRaMbLEants), yWJS044, yWJS047, yWJS067, yWJS184 and yWJS321. Among the rearranged strains analyzed, yWJS044, yWJS047 and yWJS067 were obtained from the starting strain yWJS1 after the first round of SCRaMbLE, yWJS184 was obtained from yWJS067 after the third round of SCRaMbLE, and yWJS321 was obtained from yWJS184 , the rearrangement strain obtained after the fifth round of SCRaMbLE.

[0088] image 3 The middle is a boxplot based on the WGS results, showing the coverage depth of different chromosomes, representing the copy number of different chromosomes, showing that the haploid S. cerevisiae synthetic ring V chromosome SCRaMbLE will lead to chromosome aneuploidy. Strains yWJS044 (aneuploid chromosomes: I, III,...

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Abstract

The invention especially relates to a generation method for the variation of the chromosome number of Saccharomyces cerevisiae, belonging to the field of synthetic biology. According to the invention,in virtue of the characteristics of genome rearrangement of Saccharomyces cerevisiae and through genome rearrangement of the ring chromosomes of Saccharomyces cerevisiae, an aneuploid Saccharomyces cerevisiae strain with chromosome number variation is obtained, the continuous variation of the circular chromosome copy number of Saccharomyces cerevisiae is realized, a Saccharomyces cerevisiae strain with continuously evolved phenotypes is obtained, and correlation between structural variation and phenotype enhancement is revealed. According to the invention, the variation of the chromosome number, the copy number and the structure of Saccharomyces cerevisiae is realized and the structural variation is revealed by using a chromosome rearrangement method, and a novel research means is provided for obtaining aneuploid Saccharomyces cerevisiae strains and studying the relationship between chromosome structure variation and functions.

Description

technical field [0001] The invention relates to the field of synthetic biology, in particular to gene elements and their application in chromosome number, copy number and structural variation of Saccharomyces cerevisiae. Background technique [0002] The increase and loss of the entire chromosome or chromosome fragment in the nucleus of eukaryotes will lead to the variation of chromosome number, produce aneuploid cells, and lead to the change of biological genetic material. Aneuploidy has been linked to diseases such as cancer and developmental disorders. Saccharomyces cerevisiae contains a large number of genes with homology to human genes, so it is widely used in the study of human diseases. Yeast is an important model organism for molecular genetic studies of aneuploidy. The understanding of aneuploidy has improved dramatically with the development of methods to selectively generate aneuploid yeast strains without causing other genetic changes, techniques to detect aneu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12P17/16C12R1/865
CPCC12P17/165C07K14/395
Inventor 元英进谢泽雄王娟李炳志
Owner TIANJIN UNIV
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