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Construction and application of saccharomyces cerevisiae strain for extracellular transport of vitamin D3 precursor squalene

A technology of Saccharomyces cerevisiae and recombinant Saccharomyces cerevisiae, which is applied in the field of Saccharomyces cerevisiae strain construction to achieve enhanced effects, broad application prospects, and strong production performance

Pending Publication Date: 2021-11-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the complex feedback regulation mechanism and limited storage space in S. cerevisiae cells have brought great difficulties to the further improvement of squalene production.

Method used

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  • Construction and application of saccharomyces cerevisiae strain for extracellular transport of vitamin D3 precursor squalene
  • Construction and application of saccharomyces cerevisiae strain for extracellular transport of vitamin D3 precursor squalene
  • Construction and application of saccharomyces cerevisiae strain for extracellular transport of vitamin D3 precursor squalene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Construction of Saccharomyces cerevisiae strain Y1

[0056] Specific steps are as follows:

[0057] (1) Artificially synthesized gene fragment P GPD -tHMG1-T ADH1 (The nucleotide sequence is shown in SEQ ID NO.1);

[0058] Using the Saccharomyces cerevisiae BY4741 genome as a template, the primer sequences described in Table 1 were used to amplify the gene fragment ROX1-UP with primers ROX1-UP-F and ROX1-UP-R;

[0059] The gene fragment ROX1-DOWN was amplified with primers ROX1-DOWN-F and ROX1-DOWN-R;

[0060] Using the plasmid pMHyLp-His (nucleotide sequence shown in SEQ ID NO.2) as a template, the ROX1-His fragment was amplified using primers ROX1-loxH-F and ROX1-loxH-R.

[0061] (2) The four fragments P in step (1) GPD -tHMG1-T ADH1 , ROX1-UP, ROX1-DOWN, and ROX1-His were fusion-PCRed by PCR, and the correct band obtained by running the gel was recovered by cutting the gel to obtain the fusion gene fragment ΔROX1-P GPD -tHMG1-T ADH1 .

[0062] (3)...

Embodiment 2

[0064] Example 2: Construction of Saccharomyces cerevisiae strain Y2

[0065] (1) Artificially synthesized gene fragment P TEF1 -IDI-T CYC1 (The nucleotide sequence is shown in SEQ ID NO.4);

[0066] Using the Saccharomyces cerevisiae BY4741 genome as a template, the primer sequences shown in Table 1 were used to amplify the gene fragment 911b-UP with primers 911b-UP-F and 911b-UP-R;

[0067] Amplify the gene fragment 911b-DOWN with primers 911b-DOWN-F and 911b-DOWN-R;

[0068] Using the plasmid pMHyLp-His as a template, the INO2-His fragment was amplified with primers IDI-loxH-F and IDI-loxH-R.

[0069] (2) The four fragments P in step (1) TEF1 -IDI-T CYC1 , 911b-UP, 911b-DOWN, IDI-His use PCR for fusion PCR, and the correct band obtained by running the gel is recovered by cutting the gel to obtain the fusion gene fragment 911b-P TEF1 -IDI-T CYC1 .

[0070](3) Transform the gene fragment in step (2) into the competent Y1 strain prepared in Example 1, culture it on the...

Embodiment 3

[0072] Example 3: Construction of Saccharomyces cerevisiae strain Y3

[0073] Specific steps are as follows:

[0074] (1) Artificially synthesized gene fragment P GPD -tHMG1-T ADH1 -P TEF1 -ERG20-T CYC1 (The nucleotide sequence is shown in SEQ ID NO.5);

[0075] Using the Saccharomyces cerevisiae BY4741 genome as a template, the primer sequences described in Table 1 were used to amplify the gene fragment ERG20-UP with primers ERG20-UP-F and ERG20-UP-R;

[0076] The gene fragment ERG20-DOWN was amplified with primers ERG20-DOWN-F and ERG20-DOWN-R;

[0077] Using the plasmid pMHyLp-His as a template, the ERG20-His fragment was amplified with primers ERG20-loxH-F and ERG20-loxH-R.

[0078] (2) The four fragments P in step (1) GPD -tHMG1-T ADH1 -P TEF1 -ERG20-T CYC1 -P PGK , ERG20-UP, ERG20-DOWN, and ERG20-His use PCR for fusion PCR, and the correct bands obtained by running the gel are recovered by cutting the gel to obtain the fusion gene fragment P GPD -tHMG1-T ADH...

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Abstract

The invention discloses construction and application of a saccharomyces cerevisiae strain for extracellular transport of vitamin D3 precursor squalene, and belongs to the technical field of fermentation engineering. The construction comprises the following steps of: firstly, constructing a recombinant saccharomyces cerevisiae strain Z1O3 for high yield of squalene, wherein the recombinant saccharomyces cerevisiae intensively expresses 3-hydroxy-3-methylglutaryl coenzyme A reductase, isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthase, and related genes INO2, Pdr5 protein and Osh3 protein of endoplasmic reticulum size regulating factors; knocking out ROX1 gene; when the strain is subjected to shake flask fermentation, the content of squalene in extracellular dodecane of the strain reaches 893.58mg / L; and when the strain is cultured in a fermentation tank, the content of squalene in extracellular dodecane of the strain can reach 6995.81mg / L. The recombinant strain constructed by the invention not only has the ability of extracellular transport of squalene, but also has stronger squalene production performance, and has broad application prospects.

Description

technical field [0001] The invention relates to the construction and application of a Saccharomyces cerevisiae strain for extracellular transport of vitamin D3 precursor squalene and belongs to the technical field of fermentation engineering. Background technique [0002] Saccharomyces cerevisiae (S.cerevisiae) is one of the most widely used model microorganisms in the fermentation industry, with many advantages such as clear genetic background and convenient gene manipulation. The naturally occurring mevalonate pathway (MVA pathway) and post-squalene pathway (post-squalene pathway) in Saccharomyces cerevisiae can synthesize squalene, lanosterol, ergosterol, etc. It can also realize the synthesis of vitamin D3 precursor (7-DHC) by introducing exogenous genes. [0003] Squalene is an important platform compound in the cells of S. cerevisiae, which is closely related to the synthesis of lipids, and is also a key precursor and limiting factor for the synthesis of vitamin D3. ...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/53C12N15/61C12N15/54C12N15/31C12P5/02C12R1/865
CPCC12N9/0006C12N9/90C12N9/1085C07K14/395C12N15/81C12P5/007C12Y101/01088C12Y503/03002C12Y205/01068C12Y205/0101C12Y205/01092Y02A50/30
Inventor 刘龙陈坚吕雪芹堵国成李江华刘延峰刘家恒金柯
Owner JIANGNAN UNIV
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