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Plasmid toolkit for multi-copy integration of saccharomyces cerevisiae

A technology of Saccharomyces cerevisiae and integration plasmid, applied in the field of gene and metabolic engineering, can solve the problems of limited integration site application, unknown synergistic integration ability, insufficient screening tags, etc., and achieve the effect of high-efficiency expression

Pending Publication Date: 2021-09-17
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the current integrated expression based on the Ty site has many defects, such as insufficient screening tags and low copy number of the obtained transformant; the integrated expression frame uses the same promoter, the same promoter and terminator and other expression elements, Increases the probability of homologous recombination; the ability to coordinate integration between different Ty sites is unknown, etc., limiting the application of such integration sites

Method used

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  • Plasmid toolkit for multi-copy integration of saccharomyces cerevisiae
  • Plasmid toolkit for multi-copy integration of saccharomyces cerevisiae
  • Plasmid toolkit for multi-copy integration of saccharomyces cerevisiae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1: construct the plasmid expression kit of multiple copies

[0064] In the present invention, Gibson assembly technology (Gibson assembly) is used as the assembly means, and the assembly of all sequences is seamless cloning. The corresponding nucleotide sequence is amplified by PCR, and the sequences contain about 20-30bp homology arms, and all plasmid expression kits can be obtained after Gibson assembly.

[0065] (1) Construction of multi-copy integrated screening gene expression cassette combination

[0066] Select 10 combinations of screening gene expression cassettes for multi-copy integration (Marker genes, as shown in Table 2 and figure 1 shown in pT0~pT9), including 10 weak promoter sequences, 10 screening gene sequences with degradation tags deg, and terminator T TDH3 , the nucleotide sequence is SEQ ID NO.43.

[0067] The 10 weak promoters were P ADE6 ,P LEU2 ,P URA3 ,P PMA1 ,P ZWF1 ,P ARO7 ,P PYC1 ,P ADE3 ,P YEF3 ,P ERG1 , the correspo...

Embodiment 2

[0090] Example 2: Verification of integration ability of multi-copy integration plasmid tool

[0091] Use primers Ty11-inte-up / down to amplify plasmids pcT110~pcT119, use primers Ty12-inte-up / down to amplify plasmids pcT120~pcT129, use primers Ty2-inte-up / down to amplify pcT20~pcT29 1. Use primers Ty3-inte-up / down to amplify plasmids pcT30-pcT39 respectively, and use primers Ty4-inte-up / down to amplify plasmids pcT40-pcT49 respectively, to obtain the integrated expression frame part of the vector respectively. After recovering and purifying the PCR product, use the high-efficiency transformation method of Saccharomyces cerevisiae (see Gietz, R.D. for specific transformation methods; Schiestl, R.H., High-efficiency yeast transformation using the LiAc / SS carrier DNA / PEG method [J]. Nat Protoc 2007, 2(1 ), 31-4.) into Saccharomyces cerevisiae strains.

[0092] Transform into strain C800 when the screening tags are TRP1, LEU2, URA3, HIS5, natMX, hphMX, patMX, bleMX. When the scr...

Embodiment 3

[0096] Embodiment 3: the application of multi-copy integration plasmid kit

[0097] The synthesis process of Taxusin involves the introduction and overexpression of multiple genes, so it was used to verify the performance of the multi-copy integration plasmid kit. The synthetic pathway of pacitaxel is as follows Figure 4 As shown, the first module is from glucose to p-coumaric acid, the second module is from p-coumaric acid to naringenin, and the third module is from naringenin to taxanthin. The multi-copy integrated plasmid toolkit with significantly increased copy number in Example 2 was selected and applied to the production of pacitaxel or related intermediate products.

[0098] 1. Construction of single Ty site integrated recombinant bacteria

[0099] Using primers Ty4-inte-up / Ty4-inte-down to amplify pcfB4-47LL and pcfB4-P05m4 respectively, the PCR products were efficiently transformed by S. C805. Strain C8011 integrated the genes related to the synthesis of p-couma...

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Abstract

The invention discloses a group of plasmid toolkits for multi-copy integration of saccharomyces cerevisiae, and belongs to the field of gene and metabolic engineering. Theplasmid toolkit for the multi-copy integration constructed and verified by the invention can realize multi-copy, stable and integrated expression of a plurality of exogenous genes on a saccharomyces cerevisiae genome through one-time transformation. In addition, by selecting different screening tags at different Ty sites, multi-batch, multi-gene, stable and integrated expression of exogenous genes can be realized.

Description

technical field [0001] The invention relates to a group of plasmid kits for multi-copy integration of Saccharomyces cerevisiae, belonging to the field of gene and metabolic engineering. Background technique [0002] As a model strain of eukaryotes, Saccharomyces cerevisiae is often used to introduce heterologous pathways and synthesize high value-added products. These pathways generally involve several to dozens of genes, but the co-expression of multiple genes in Saccharomyces cerevisiae requires multiple integrations, which is inefficient, and the integration sites and screening tags cannot meet the subsequent integration operations. Therefore, there is an urgent need for an efficient, high-copy and stable insertion site to integrate genes to be overexpressed into these sites to achieve high-efficiency expression of heterologous genes. [0003] There are many kinds of long terminal repeats (LTR) in the genome of Saccharomyces cerevisiae, and one type of LTR is a transposo...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N1/19C12P17/06C12R1/865
CPCC12N15/81C07K14/395C12P17/06
Inventor 周景文高松曾伟主陈坚沐万孟
Owner JIANGNAN UNIV
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