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Construction method and application of saccharomyces cerevisiae genetically engineered bacterium with high yield of 3-methylthio-propanol

A technology of Saccharomyces cerevisiae and methylthiopropanol, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve problems such as toxic by-products and serious environmental pollution, and achieve the effect of increasing the synthesis amount

Inactive Publication Date: 2015-07-01
BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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Problems solved by technology

Although the chemical synthesis of aroma substances has the advantages of low cost and high output, there are disadvantages such as serious environmental pollution and the production of toxic by-products.

Method used

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  • Construction method and application of saccharomyces cerevisiae genetically engineered bacterium with high yield of 3-methylthio-propanol
  • Construction method and application of saccharomyces cerevisiae genetically engineered bacterium with high yield of 3-methylthio-propanol
  • Construction method and application of saccharomyces cerevisiae genetically engineered bacterium with high yield of 3-methylthio-propanol

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Embodiment Construction

[0038] 1. Cloning of ARO8 and ARO10 genes

[0039] 1.1 Extraction of Saccharomyces cerevisiae S288c genomic DNA

[0040] The freshly activated S. cerevisiae S288c strain was inoculated into YPD liquid medium, placed in a shaker at 30°C, 220rpm and shaken overnight.

[0041] (1) Take 1 mL of bacterial liquid into a 1.5 mL centrifuge tube, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the supernatant;

[0042] (2) Add 600 μL of sorbitol Buffer to the cell pellet, add 100 μL of 100 mg / mL helicase, mix well, place in a water bath shaker at 30°C at 220 rpm for 90 minutes;

[0043] (3) YeastDNA Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used to extract the genome of Saccharomyces cerevisiae.

[0044] 1.2 Cloning of ARO8 gene

[0045] Using Saccharomyces cerevisiae S288c genomic DNA as a template, specific primers were designed to amplify the ARO8 gene fragment by PCR.

[0046] ARO8 gene primer sequence:

[0047] Upstream primer: 5'-CGGGAT...

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Abstract

The invention relates to a recombinant saccharomyces cerevisiae engineering strain and a preparation method thereof. The recombinant saccharomyces cerevisiae engineering strain carries a transaminase gene expression carrier and a decarboxylase gene expression carrier which are connected in series. The preparation method of the saccharomyces cerevisiae engineering strain comprises the following steps: 1) respectively amplifying a transaminase gene fragment and a decarboxylase gene fragment from a saccharomyces cerevisiae genome; 2) designing a specific restriction enzyme digestion site, and connecting the specific restriction enzyme digestion site on escherichia coli-saccharomyces cerevisiae shuttle plasmid vector pYES-pgk in series; and 3) transforming recombinant plasmid into saccharomyces cerevisiae by adopting a PEG / LiAc method. The prepared recombinant saccharomyces cerevisiae engineering strain can be used for obviously increasing synthetic amount of a flavour component 3-methylthio-propanol and can be applied to the industries of food, medicines and daily chemicals.

Description

technical field [0001] The invention relates to a construction method and application of Saccharomyces cerevisiae genetically engineered bacteria with a high yield of 3-methylthiopropanol, which belongs to the field of biological production of food flavors, and the prepared natural 3-methylthiopropanol flavors can be used for the preparation of various food flavors , Widely used in food, medicine, daily chemical and other industries. Background technique [0002] 3-Methylthio-1-propanol (3-Methylthio-1-propanol) is an important food spice. 3-Methylthiopropanol is a volatile flavor substance with a low aroma threshold. It presents meaty, boiled potato and vegetable flavors. It is naturally present in vegetables, fruits, wine and a variety of fermented foods and seasonings. It is an important flavor substance of sesame-flavored liquor, red wine and soy sauce. 3-Methylthiopropanol and its derivatives (3-Methylthiopropionaldehyde, Ethyl 3-Methylthiopropionate and Methyl 3-Meth...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/11C12R1/865
CPCC12N9/1096C12N9/88C12N15/81C12Y206/01
Inventor 尹胜王成涛孙宝国
Owner BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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