High-salt-tolerant saccharomyces cerevisiae strain as well as construction method and application thereof

A technology of Saccharomyces cerevisiae strain and construction method, applied in the field of Saccharomyces cerevisiae strains, can solve problems such as the need to improve salt tolerance, and achieve the effects of shortening fermentation period, high salt tolerance, and increasing unsaturated fatty acid content

Pending Publication Date: 2020-06-02
JIANGXI SCI & TECH NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims at the technical defects of the prior art, and provides a high-salt-tolerant Saccharomyces cerevisiae strain, its construction metho

Method used

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  • High-salt-tolerant saccharomyces cerevisiae strain as well as construction method and application thereof
  • High-salt-tolerant saccharomyces cerevisiae strain as well as construction method and application thereof
  • High-salt-tolerant saccharomyces cerevisiae strain as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment one (construction and identification of recombinant plasmid)

[0034] 1. Amplification of the target gene

[0035] Aspergillus oryzae 3042 was cultured at 30°C for 3 days, then sampled, immediately frozen in liquid nitrogen, and stored in a -70°C refrigerator; total RNA was extracted using TRIzol Reagent (Invitrogen, USA), and the operation method was according to the kit provided by the company Instructions are carried out. After extraction, DNase I (RNase-free) from Fermentas Company was used to remove the remaining genomic DNA in the total RNA. The total RNA was then reverse-transcribed to synthesize the first strand of cDNA. The specific method was operated according to the instructions of the TIANScript cDNA First Strand Synthesis Kit (Beijing Tiangen Biotechnology Co., Ltd.). Primer amplification is designed according to the target gene sequence, and specific fragments are recovered.

[0036] The primers are as follows (the underlined part is the res...

Embodiment example 2

[0043] Implementation Case 2 (Acquisition of Recombinant Bacteria)

[0044] 1. Transformation of Saccharomyces cerevisiae

[0045] Take yeast competent cells stored at -80°C and thaw on ice. Prepare the premix: PEG240μL, 1M LiAc36μL, Carrier DNA 10μL, target plasmid 5μL, add sterile deionized water to make up to 360μL. Add the premix solution containing the target plasmid into the competent cells, and repeatedly blow and aspirate the sediment to completely suspend the yeast cells in the premix solution. Incubate in a water bath at 30°C for 30 minutes, mix well every 10 minutes; heat shock in a water bath at 42°C for 30 minutes, mix well every 10 minutes. Centrifuge at 12000rpm for 1min, remove the supernatant, add 100μL of sterile deionized water to suspend the precipitate, spread the bacterial solution on the SD-Ura medium plate, and incubate at 30°C for 2 days.

[0046] 2. Identification and storage of positive clones

[0047] Positive clones were identified by bacterial...

Embodiment example 3

[0048] Implementation case three (26S rRNA identification experiment of Saccharomyces cerevisiae strain of the present invention)

[0049] Yeast genomic DNA was extracted and purified using the method of Sambrook et al. (1989). Using the extracted and detected qualified genomic DNA as a template, the primer pair NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL4 (5'-GGTCCGTGTTTCAAGACGG-3') were selected for PCR amplification of the 26S rRNA gene sequence according to Solieri et al. ( 2007), the gene sequence sequencing was completed by Shanghai Sangon Sequencing Company.

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Abstract

The invention provides a high-salt-tolerant saccharomyces cerevisiae strain as well as a construction method and application thereof. In the technical solution, an oleic acid synthase encoding gene ofaspergillus oryzae 3042 is extracted and transformed into a natural yeast strain through a vector, and the obtained recombinant strain individually expresses oleic acid synthase derived from the aspergillus oryzae 3042; based on the morphological characteristics and search results of a 26S rRNA gene sequence in Genbank, the strain is identified as saccharomyces cerevisiae; experimental verification shows that the strain provided by the invention can grow and reproduce in a high-salt environment of soy sauce brewing, and has a higher content of unsaturated fatty acids compared with current strains; and the strain provided by the invention is used as a soy sauce brewing strain, the smooth progress of the fermentation process under high-salt conditions can be ensured, the fermentation cyclecan be shortened to a certain extent, and the product has a higher content of unsaturated fatty acids, and is an excellent strain for soy sauce brewing.

Description

technical field [0001] The invention relates to the technical field of industrial microbes, in particular to a high-salt-tolerant Saccharomyces cerevisiae strain, its construction method and application. Background technique [0002] Soy sauce is one of the most important traditional condiments in my country. Aspergillus and yeast are the main fermentation microorganisms in soy sauce brewing. The proteases of these microorganisms can degrade the protein in the raw materials into small molecules and polypeptides that are easily absorbed by the human body. At the same time, the metabolism of these microorganisms Products, such as small molecule alcohols, aldehydes, acids, esters, unsaturated fatty acids, phenols, etc., can also provide flavor for soy sauce. [0003] According to the different brewing and fermentation processes of soy sauce, it can be divided into solid salt-free, low-salt solid state and high-salt dilute state. The products provided by solid-state salt-free an...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81A23L27/50C12R1/865
CPCC12N9/88C12Y402/01053C12N15/81A23L27/50
Inventor 贺斌刘华曾斌涂雅怡
Owner JIANGXI SCI & TECH NORMAL UNIV
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