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Yeast Engineering Strain Transformed by Chromosomal Fusion

A technology for yeast engineering and engineering strains, applied in genetic engineering, stable introduction of foreign DNA into chromosomes, microorganism-based methods, etc., and can solve problems such as limiting the application of heterologous gene expression

Active Publication Date: 2021-06-08
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These deficiencies in S. cerevisiae limit its utility as a host for heterologous gene expression and large DNA cloning

Method used

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  • Yeast Engineering Strain Transformed by Chromosomal Fusion
  • Yeast Engineering Strain Transformed by Chromosomal Fusion
  • Yeast Engineering Strain Transformed by Chromosomal Fusion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0212] Example 1 , strain construction

[0213] The starting strain of a series of chromosomally fused engineering strains constructed by the present invention is the derivative strain BY4742 of Saccharomyces cerevisiae wild-type strain S288C, and its genotype is MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 (Brachmann, C.B., etc. (1998); Yeast, 14, 115- 132).

[0214] There are 13 major repeat sequences in the proximal telomeric region of the 16 natural chromosomes of strain BY4742 (Table 1), of which repeat sequences 1, 7, 9, and 10 are 3 copies, and the remaining 11 repeat sequences are 2 copies. The length of these repeated sequences is 2.7-26.2kb, and they tend to become hotspots of chromosome homologous recombination, thereby affecting the stability of the entire genome, and also interfere with the site-directed homologous recombination of chromosome fusion. In order to eliminate the main repeat sequence near the telomere, the inventors deleted a total of 19 repeat sequences, incl...

Embodiment 2

[0232] Example 2 , strain performance

[0233] Strain growth

[0234] A series of chromosomal fusion engineered strains of Saccharomyces cerevisiae constructed by the invention have good growth. The one-by-one fusion of linear natural chromosomes did not affect the growth of the strain. Figure 5 The growth curve of the strain SY11-SY13 ( Figure 5 , A) and SY14 ( Figure 5 , B) The growth was almost identical to that of the wild-type starting strain BY4742. The circularization of the chromosome made the growth of the strain SY15 slightly slower than that of the wild-type strain BY4742 ( Figure 5 , B).

Embodiment 3

[0235] Example 3 , genome stability

[0236] A series of chromosomal fusion engineered strains of Saccharomyces cerevisiae constructed by the invention have genetically stable genomes. Image 6 Shown are the enzyme digestion verification pictures of the genomes of strains SY14 and SY15 after 0 generation and continuous growth of 100 generations. The chromosome of strain SY14 can theoretically cut out 23 bands with restriction endonuclease FseI, which are 1569, 1446, 1125, 1050, 892, 891, 778, 587, 548, 414, 412, 381, 363, 318, 258 , 210, 141, 133, 82, 78, 27, 25.7, and 0.9 kb. The chromosome of the strain SY15 is derived from the circularization of the chromosome of SY14, so comparing the FseI restriction map of SY15 with that of SY14, two differential bands of 1125 and 82 kb will be reduced. These two bands were merged into one in SY15, that is, a new 1193kb differential band was added.

[0237] The de novo sequencing whole genome sequence of strain SY14 is shown in SEQ ...

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Abstract

The invention relates to a yeast engineering strain transformed by chromosomal fusion. The present inventors artificially fused 16 natural chromosomes of yeast to obtain a series of chromosomally fused strains, including 16 linear or circular chromosomes fused into one. The yeast engineering strain of the invention can be used as a host for eukaryotic heterologous gene expression and large-segment DNA cloning.

Description

technical field [0001] The invention belongs to the fields of microbial synthetic biology, genome engineering and molecular biology, and more specifically, the invention relates to yeast engineering strains transformed by chromosome fusion. Background technique [0002] Saccharomyces cerevisiae is a single-celled eukaryote and the first eukaryotic model organism to be sequenced. Its genome size is 12Mb and contains 16 linear chromosomes. Saccharomyces cerevisiae has been used in alcohol brewing and food fermentation for a long time. Studies in many aspects such as genetics, molecular biology and physiology have shown that wild-type strains of Saccharomyces cerevisiae are safe for humans. Saccharomyces cerevisiae is easy to cultivate, has a clear genetic background, and relatively simple genetic operations. In addition, it has a eukaryotic protein post-translational processing and modification system, so it has become a commonly used host for heterologous gene expression in h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N15/113C12R1/865
CPCC12N15/113C12N15/905C12N2310/10
Inventor 覃重军邵洋洋薛小莉鲁宁
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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