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Saccharomyces cerevisiae strain for synthesizing squalene and application thereof

A technology of Saccharomyces cerevisiae and squalene, applied in the field of genetic engineering, can solve the problems of limited output and long fermentation cycle, and achieve the effect of strengthening metabolic flow

Active Publication Date: 2021-08-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are documents in the prior art to carry out genetic engineering transformation to Saccharomyces cerevisiae to improve the scheme of squalene production, but there are still problems such as long fermentation period and limited output

Method used

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  • Saccharomyces cerevisiae strain for synthesizing squalene and application thereof
  • Saccharomyces cerevisiae strain for synthesizing squalene and application thereof
  • Saccharomyces cerevisiae strain for synthesizing squalene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Single-copy site integration of different copy numbers tHMG1 and IDI1

[0064] Select the sites ARO10, EXG1, and SPR1 of Saccharomyces cerevisiae C800, and increase tHMG1 (corresponding nucleotide sequence such as the sequence table SEQ ID NO.9) and IDI1 (the corresponding nucleotide sequence is shown in the sequence table as SEQ ID NO.10), so that the gene tHMG1 and IDI1 copies of the constructed Saccharomyces cerevisiae engineered bacteria are respectively 1, 2 or 3 copies. The strains integrating different copy numbers were fermented in YPD medium at 30°C and 220rpm for 96h to obtain a positive correlation between squalene production and copy number, as shown in figure 1 . The squalene production of strain CP02 with 1 copy of tHMG1-IDI1 was 121.2 mg / L; the squalene production of strain CP05 with 2 copies of tHMG1-IDI1 was 252.1 mg / L; 3 copies of tHMG1 - The squalene production of strain CP12 of IDI1 was 426.0 mg / L.

Embodiment 2

[0065] Example 2 Integration of multiple copy sites of tHMG1 and IDI1

[0066] In order to obtain more copy numbers, the multi-copy site Ty2 transposon was selected for the integration of tHMG1 and IDI1. The Ty2 transposon structure searched in SGD is as follows figure 2 (SGD:S000007168), the upstream and downstream homology arms are designed at the LTR site. LEU was added as a screening marker. In order to increase the integrated copy number and increase the LEU degradation tag, the genes tHMG1 and IDI1 were respectively amplified from the S. cerevisiae 800 genome with primers tHMG1-F / tHMG1-R and IDI1-F / IDI1-R. Primers Ty2-armup-F / Ty2-armup-R and Ty2-armdown-F / Ty2-armdown-F respectively amplify the upstream and downstream homology arms of the Ty2 transposon, using pY15-F / pY15-R from a commercial plasmid The plasmid backbone pY15-1 was amplified from pY15, and the above fragments (genes tHMG1 and IDI1, upstream and downstream homology arms of Ty2 transposon) were assembled u...

Embodiment 3

[0070] Example 3 Heterologous expression of NADH-dependent HMG1 regulates intracellular redox balance

[0071] The gene NADH-HMG1 derived from S. pomeroyi was synthesized (the nucleotide sequence is shown in SEQ ID NO.1, and the corresponding amino acid sequence is shown in SEQ ID NO.2), and the plasmid pUC57- NADH-HMG1, using primers NADH-HMG1-F / NADH-HMG1-R to amplify the NADH-HMG1 fragment, using primers GAL-ARMUP-F / GAL-ARMUP-R and GAL-ARMDOWN-F / GAL-ARMDOWN-R The upper and lower homology arms of the GAL site were amplified respectively, and the upper and lower homology arms of the GAL site were combined with the NADH-HMG1 fragment sgRNA using the yeast transformation kit Frozen-EZ YeastTransformation II to transform the fragment into the one constructed in Example 2 In the strain XSQ10-1 cells, spread on SD-HIS-TRP screening solid medium, culture at 30°C for 3 days to obtain strain XSQ11, pick a single colony of XSQ11 in YPD medium, culture at 30°C for 96h, squalene Compare...

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Abstract

The invention discloses a saccharomyces cerevisiae strain for synthesizing squalene and an application of the saccharomyces cerevisiae strain, and belongs to the technical field of genetic engineering. According to the invention, tHMG1 and IDI1 are integrated at a multi-copy site Ty2 of saccharomyces cerevisiae, so that synthesis and accumulation of squalene are remarkably promoted, NADH-dependent HMG1 is heterologously expressed, the redox level in yeast cells is balanced, a promoter HXT1 with a remarkable inhibition effect on ERG1 transcription is further replaced, conversion of squalene to the downstream is hindered, ACL1 and ACL2 are heterologously expressed, PEX11 and FOX3 are over-expressed, accumulation of a squalene synthesis precursor acetyl coenzyme A in cytoplasm is enhanced through beta oxidation, accumulation of squalene is further promoted, and the squalene yield can reach 1253.4 mg / L after saccharomyces cerevisiae is fermented for 96 h.

Description

technical field [0001] The invention relates to a Saccharomyces cerevisiae strain for synthesizing squalene and an application thereof, belonging to the technical field of genetic engineering. Background technique [0002] Squalene (squalene) is an important terpenoid compound, which is the precursor of many bioactive compounds, such as steroids and hopene. At the same time, squalene has many values, such as being used in the cosmetic industry as a moisturizing ingredient, due to His antioxidant properties can be used as a moisturizer, and squalene is also beneficial to human health, including anti-tumor, anti-fungal / fungal, enhancing the body's immunity, and lowering cholesterol, such as anti-inflammatory, anti-aging, anti-oxidation, etc. Squalene is also widely used to make stable emulsions, such as vaccines, drugs and other medicinal substrates. The traditional method extracts from shark liver by illegal means or inefficiently extracts from plants, but in view of its lim...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12P5/02C12R1/865
CPCC12P5/026C12N9/88C12N9/0006C12Y401/03C12Y101/01088
Inventor 曾伟主周景文夏路陈坚堵国成
Owner JIANGNAN UNIV
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