High yield tetramethylpyrazine saccharomyces cerevisiae and construction method thereof

A technology of tetramethylpyrazine and Saccharomyces cerevisiae, which is applied in the field of bioengineering, can solve problems such as application limitations, and achieve the effects of not being easy to reverse mutations, increasing production, and ensuring safety

Inactive Publication Date: 2017-07-07
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the actual liquor fermentation process is carried out in an anaerobic and high acidity environment. After these two types of bacteria enter the fermentation cellar, their growth and metabolism quickly stop, which greatly limits their application.
Only by fermenting and cultivating these two types of bacteria outside the fermentation cellar can a higher TTMP yield be generated, so these methods are generally only suitable for the production of Maotai-flavored liquor and sesame-flavored liquor with accumulation and cultivation outside the cellar

Method used

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  • High yield tetramethylpyrazine saccharomyces cerevisiae and construction method thereof
  • High yield tetramethylpyrazine saccharomyces cerevisiae and construction method thereof
  • High yield tetramethylpyrazine saccharomyces cerevisiae and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Construction of Knockout BDH1 Gene and Overexpression BDH2 Gene Saccharomyces cerevisiae Strain

[0038]The starting strain used in this example is Saccharomyces cerevisiae CICC32315, the Escherichia coli DH5a is purchased from Takara Company, and the YEPD medium is a universal complete medium.

[0039] The construction process of the recombinant plasmid Yep-PB2K is as follows: figure 1 shown. The PGK1 promoter and terminator gene on the pPGK1 plasmid were excised and then connected to the Yep352 plasmid to obtain Yep-PGK1; the gene encoding diacetyl reductase BDH2 derived from Saccharomyces cerevisiae obtained by PCR was inserted into the PGK1 promoter and Between the terminators, Yep-PGK1-BDH2 was obtained; the KanMX resistance gene derived from pUG6 was connected to obtain the plasmid Yep-PGK1-BDH2-Kan, which was named as plasmid Yep-PB2K.

[0040] BDH1 recombination process such as figure 2 shown. Using the constructed vector plasmid Yep-PB2K as a te...

Embodiment 2

[0046] Example 2: Construction of Saccharomyces cerevisiae strain with complete deletion of BDH1 gene

[0047] According to the method in Example 1, the following scheme was designed to obtain a recombinant strain of Saccharomyces cerevisiae that knocked out the BDH1 gene. The starting strain used in this example was also Saccharomyces cerevisiae CICC32315, and the YEPD medium was a universal complete medium.

[0048] Using the KanMX resistance gene on the pUG6 plasmid as a template, the nucleotide sequence is shown in SEQ NO: 13 in the nucleotide sequence list. Using long primers B1K-U (SEQ NO: 14) and B1K-D (SEQ NO: 15) for PCR amplification to obtain homologous fragments B1KA and B1KB at both ends of the 2,3-butanediol dehydrogenase BDH1 gene The recombinant gene fragment, the long primer sequence for sequence fragment amplification is:

[0049] B1K-U: 5'-GGAACTAAAAAAGTTTTTAATTAATTATGAGAGCTTTGGCATATTTCAACAGCTGAAGCTTCGTACGC-3'

[0050] B1K-D: 5'-CGCGAGGGGCCCCAAATATTATTTTTG...

Embodiment 3

[0058] Example 3: Knockout of BDH1 gene and overexpression of BDH2 gene Saccharomyces cerevisiae strain fermentation experiment

[0059] (1) Corn thick mash fermentation experiments of recombinant strains and starting strains

[0060] The recombinant strain and the original strain were subjected to corn thick mash fermentation experiments at the same time, and the fermentation process route was: corn flour→soaking→liquefaction→saccharification→cooling→inoculation→fermentation→steaming wine→measurement indicators;

[0061] Process conditions:

[0062] Soaking conditions: 60-70°C, soaking for 20 minutes; liquefaction conditions: 85-90°C, adding high-temperature resistant α-amylase, liquefying for 90 minutes; saccharification conditions: 55-60°C, adding glucoamylase, saccharifying for 20 minutes.

[0063] Determination of strain CO after fermentation 2 Fermentation performance indicators such as cumulative discharge, alcohol content and residual reducing sugar, the results are ...

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Abstract

The invention discloses a high yield tetramethylpyrazine saccharomyces cerevisiae strain and belongs to the technical field of bioengineering technology. The saccharomyces cerevisiae strain having similar fermenting property of a parent strain but being high in yield of tetramethylpyrazine is bred. Compared with the parent strain, the other fermenting properties of the obtained saccharomyces cerevisiae strain are not affected in the liquor fermentation test, the content of the tetramethylpyrazine is remarkably improved by increasing the content of a precusor substance acetoin, the content of the acetoin is 880.82 mg/L and is ten times that of the original strain, and the content of tetramethylpyrazine is 55.53 mg/L and is 4 times that of the original strain. The saccharomyces cerevisiae strain remarkably improves the yield of the tetramethylpyrazine, meets the higher requirements on yeasts in the related fields of white liquors, and has a broad application prospect.

Description

[0001] Technical field: [0002] The invention belongs to the technical field of bioengineering and relates to the breeding of industrial microorganisms, in particular to a Saccharomyces cerevisiae with high tetramethylpyrazine production and a construction method thereof. [0003] Background technique: [0004] In recent years, people not only pay attention to the taste and quality of fermented food, but also pay great attention to its health effects. Research has found that traditional Chinese fermented food liquor, vinegar, and soy sauce all contain certain pyrazines, which not only have aromatic properties, but also have the functions of dilating blood vessels, improving blood circulation, and protecting the liver (preventing alcohol from damaging the gastric mucosa and liver) and other functions (Winemaking Science and Technology, 2013 (9): 1-6), and the content of tetramethylpyrazine is the highest. In 2006, at the first Chinese Baijiu and Health Symposium, Mr. Wu Jianfe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12P17/12C12G3/02C12Q1/68C12N15/11C12R1/865
CPCC12G3/02C12N9/0006C12P17/12C12Q1/6895C12Y101/01004C12Y101/01076C12Y101/01303C12Y101/01304
Inventor 肖冬光陈诗佳张翠英郭学武陈叶福杜丽平李维董健
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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