Saccharomyces cerevisiae strain for producing 7-dehydrocholesterol and construction method

A technology for dehydrocholesterol and yeast, applied in microorganism-based methods, biochemical equipment and methods, botanical equipment and methods, etc., can solve the problem of unsatisfactory source and yield, many reaction steps, and complicated by-product removal process and other problems, to achieve the effect of green cleaning and making up for defects in the production process

Inactive Publication Date: 2013-09-04
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The consumption of 7-dehydrocholesterol is huge, but its source and production cannot meet the current growing demand
However, due to the long chemical process, many reaction steps, low yield,

Method used

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  • Saccharomyces cerevisiae strain for producing 7-dehydrocholesterol and construction method
  • Saccharomyces cerevisiae strain for producing 7-dehydrocholesterol and construction method
  • Saccharomyces cerevisiae strain for producing 7-dehydrocholesterol and construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Acquisition of genes related to 7-dehydrocholesterol biosynthesis pathway in yeast

[0041] A, acquisition of tHMGR gene (yeast truncated HMG-CoA reductase gene)

[0042] Primers were designed according to the yeast HMG-CoA reductase gene sequence, SEQ ID NO:1tHMGR-U:5'-GGAATTCGCAGGCACGTCTAGAATGGACCAATTGGT-3' and SEQ ID NO:2tHMGR-D:5'-GCGACTAGTGTTAGGATTTAATGCAGGTGACGG-3', based on the genome of Saccharomyces cerevisiae BY4742 strain As a template, use fast pfu enzyme for PCR (95°C, 2min; 95°C, 20s, 63°C, 30s, 72°C, 2min, 30cycles; 72°C, 5min; 4°C, +∞) amplification to obtain a 1578bp fragment. Cloned into the pSB1A2 vector and sequenced, it was confirmed that no mutation occurred. The sequence of the 1578bp nucleotide fragment is shown in SEQ ID NO:3, and the encoded amino acid sequence is shown in SEQ ID NO:4.

[0043] B, the acquisition of ERG1 gene (yeast squalene epoxidase gene)

[0044]Primers were designed according to the yeast ERG1 gene sequence, S...

Embodiment 2

[0047] Embodiment 2: the construction of carrier

[0048] A, construction of carrier SyBE_000923 (expression vector of truncated HMG-CoA synthase gene and ERG1 gene)

[0049] ①The yeast constitutive promoter TDH3p, tHMGR gene, and terminator PGK1t were spliced ​​together by restriction endonuclease method to obtain a fragment containing XhoⅠ and BamHI restriction sites at both ends, and connected to the integration vector pRS403;

[0050] ②Splice the yeast constitutive promoter PGK1p, ERG1 gene, and terminator PGK1t together using the restriction endonuclease method to obtain a fragment containing two sites of ApaⅠ and SalⅠ at both ends, and connect it to the vector obtained in step ① to obtain the vector SyBE_000923 ,See figure 1 ;

[0051] The tHMGR gene is the nucleotide sequence described in SEQ ID NO:3 of the sequence listing; the ERG1 gene is the nucleotide sequence described in the sequence listing SEQ ID NO:7.

[0052] Experiments have shown that by replacing the ve...

Embodiment 3

[0058] Embodiment 3: the acquisition of yeast strain SyBE_000954

[0059] Primers were designed according to the yeast LEU gene sequence, SEQ ID NO:11LEU-U:5'-TGCTATTCCAATAGACAATAAATACCTTTTTAACATTAAGCAAGGATTTTCTTAACTTC-3'and SEQ ID NO:12LEU-D:5'-TATGATTTATTGTCTGGACAAAAGTTCTGTTTTTCCCCAATGTCTGCCCCTAAGAAGAT-3'to select Saccharomyces cerevisiae BY47 containing the selection marker gene LEU The strain genome was used as a template, and the fast pfu enzyme was used for PCR (95°C, 2min; 95°C, 20s, 62°C, 30s, 72°C, 70s, 30cycles; 72°C, 5min; 4°C, +∞) amplification to obtain 1095bp Fragment (the nucleotide sequence shown in SEQ ID NO: 13 in the Sequence Listing). The PCR product was transformed into Saccharomyces cerevisiae strain YML008C by lithium acetate method, using SD-drop solid medium (deaminated yeast nitrogen source, 6.7g / l; glucose, 20g / l; Dropout mix, 0.2%; solid supplemented with 2% agar Powder) for screening, the obtained transformants were transferred to liquid medium an...

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PUM

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Abstract

The invention discloses a saccharomyces cerevisiae strain for producing 7-dehydrocholesterol and a construction method. The construction method comprises the steps as follows: 1), construction of carriers: a carrier SyBE_000923 is constructed and a carrier SyBE_000937 is constructed; 2), a saccharomyces cerevisiae strain SyBE_000954 is obtained; and 3), the carrier SyBE_000923 and the carrier SyBE_000937 are guided into the saccharomyces cerevisiae strain SyBE_000954, so that the saccharomyces cerevisiae strain for producing 7-dehydrocholesterol is obtained. According to the saccharomyces cerevisiae strain and the construction method, a synthetic biotechnology is utilized, so that the artificially synthesis of the 7-dehydrocholesterol is realized, not only can the defects of a chemical method be made up, but also the production process is environment-friendly and clean.

Description

technical field [0001] The invention relates to a Saccharomyces cerevisiae strain and a construction method for producing 7-dehydrocholesterol. Background technique [0002] 7-Dehydrocholesterol (7-DHC) is more than vitamin D 3 It is also an important precursor for the preparation of cholesteric liquid crystal materials, and has a wide range of applications in biomedicine, electromagnetic field detection, and display. The consumption of 7-dehydrocholesterol is huge, but its source and production cannot meet the current increasing demand. The existing methods for preparing 7-dehydrocholesterol are mainly chemical synthesis methods—bromination / dehydrobromination methods and oxidation-reduction elimination methods. However, due to the long process flow of the chemical method, many reaction steps, low yield, complex by-product removal process, high energy consumption and easy to cause serious pollution to the environment, the production and application of 7-dehydrocholesterol ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/81C12N1/19C12R1/865
Inventor 元英进张莹张璐刘夺周晓
Owner TIANJIN UNIV
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