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Recombinant saccharomyces cerevisiae strain and application thereof to producing tyrosol and/or salidroside

A technology of Saccharomyces cerevisiae strain and salidroside, applied in the field of genetic engineering, can solve the problems of low yield and the like, and achieve the effects of fast reproduction, easy cultivation and convenient genetic manipulation

Active Publication Date: 2019-07-09
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the E. coli expression strain can biosynthesize tyrosol and / or salidroside, the yield is low
In terms of safety, there is a large amount of endotoxin in the cell membrane space of Escherichia coli, and a trace amount of endotoxin can cause a pyrogenic reaction in the human body, making the development of products such as tyrosol and salidroside and their derivatives that are beneficial to human health IoT platforms are challenging

Method used

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  • Recombinant saccharomyces cerevisiae strain and application thereof to producing tyrosol and/or salidroside
  • Recombinant saccharomyces cerevisiae strain and application thereof to producing tyrosol and/or salidroside
  • Recombinant saccharomyces cerevisiae strain and application thereof to producing tyrosol and/or salidroside

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preparation example Construction

[0042] In the present invention, the preparation method of the Saccharomyces cerevisiae strain M5 preferably includes: the construction method of the recombinant plasmid pESC-URA3-TEF1-AROL-TDH3-ARO7*-PGK1-ARO4* and the recombinant plasmid pESC-LEU2-TEF1-TDH3- The construction method of PcAAS-PGK1-AtUGT85A1;

[0043] The prepared recombinant plasmid pESC-URA3-TEF1-AROL-TDH3-ARO7*-PGK1-ARO4* and recombinant plasmid pESC-LEU2-TEF1-TDH3-PcAAS-PGK1-AtUGT85A1 were transformed into Saccharomyces cerevisiae according to conventional methods to form Saccharomyces cerevisiae strains -M5.

[0044] In the present invention, the construction method of the recombinant plasmid pESC-URA3-TEF1-AROL-TDH3-ARO7*-PGK1-ARO4* comprises the following steps:

[0045] I. Using yeast genomic DNA as a template, using primers ScTEF1-AvrII and ScTEF1-AtaII, PCR amplified TEF1 promoter, obtained TEF1 promoter PCR product and pESC-URA3 digested with AvrII and AtaII, connected to obtain pESC-URA3 -TEF1;

...

Embodiment 1

[0074] Construction of expression strain BY4742-M5 of Saccharomyces cerevisiae

[0075] The plasmids pESC-URA3-TEF1-AROL-TDH3-ARO7*-PGK1-ARO4*, pESC-LEU2-TEF1-TDH3-PcAAS-PGK1-AtUGT85A1 were transformed into Saccharomyces cerevisiae BY4742 strain by chemical transformation.

[0076] The transformation method is as follows: take 100 μl of competent Saccharomyces cerevisiae, centrifuge to remove the supernatant, add 240 μl of 50% PEG3350, 36 μl of 1M LiAc, 100 μg of salmon essence, pESC-URA3-TEF1-AROL-TDH3-ARO7*-PGK1 -ARO4*, pESC-LEU2-TEF1-TDH3-PcAAS-PGK1-AtUGT85A1 plasmids 10μl each, and finally make up to 360μl system with water, mix gently, incubate at 30°C for 30 minutes, then heat shock at 42°C for 40 minutes After taking it out, centrifuge to remove the supernatant, add water to mix and spread on the SC plate containing uracil and leucine auxotrophs. Uracil and leucine auxotrophs were used to screen the transformed strain carrying two expression vectors at the same time, n...

Embodiment 2

[0078] This example is used to illustrate the construction of Saccharomyces cerevisiae expression strain BY4742-IM5

[0079] (1) Use Chr-URA3-F, Chr-URA3-R, ARO4*-F, ARO4*-R, ARO7*-F, ARO7*-R, AROL-F, AROL-R as primers to amplify gene integration fragments URA3, TEF1-AROL-CYC1t, TDH3-ARO7*-TEF1tPGK1-ARO4*-ADH1t. PCR reaction system: 10 μl of 5×Q5 buffer, 5 μl of 2mM dNTP, 2.5 μl of 10 μM forward primer, 2.5 μl of 10 μM reverse primer, 1 μl of template, 0.5 μl of Q5 high-fidelity DNA polymerase, and 28.5 μl of water. The reaction program is: pre-denaturation at 98°C for 30 seconds; denaturation at 98°C for 10 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 2 minutes, 30 cycles; extension at 72°C for 2 minutes to obtain the target DNA fragment.

[0080] (2) Transfer the PCR amplified bands into Saccharomyces cerevisiae BY4742 strain by chemical transformation, the transformation method is specifically: take 100 μl of competent Saccharomyces cerevisiae, centrifu...

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Abstract

The invention provides a recombinant saccharomyces cerevisiae strain for producing tyrosol and / or salidroside and application thereof and belongs to the technical field of genetic engineering. According to the recombinant saccharomyces cerevisiae strain for producing the tyrosol and / or salidroside, applied exogenous genes comprises AROL gene, PcAAS gene, AtUgt85A1 gene, ARO4* gene and ARO7* gene,which serve as five key enzyme encoding genes during biosynthesis of the tyrosol and / or salidroside and are introduced and overexpressed in saccharomyces cerevisiae, so that metabolic flux from glucose to tyrosine can be reduced to enhance the biosynthesis of the tyrosol and / or salidroside and further to achieve construction of a route of de novo synthesis from glucose to the tyrosol and / or salidroside for the saccharomyces cerevisiae.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant brewer's yeast strain, its application and a method for producing tyrosol and / or salidroside. Background technique [0002] Rhodiola rosea is a perennial herb with a very harsh growth environment. It mainly grows in areas with high cold, dryness, strong ultraviolet radiation, and large temperature difference between day and night. It has strong environmental adaptability and vitality, and is a precious traditional Chinese medicine and Tibetan medicine. Salidroside is the main active ingredient of the plant Rhodiola rosea, which has various functions such as anti-fatigue, anti-hypoxia, anti-radiation, and improving human body functions. In addition, salidroside also has pharmacological effects such as protecting kidney function, protecting the cardiovascular system, protecting the central nervous system, and improving cognitive dysfunction. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12P7/22C12P19/44C12R1/865
CPCC12N9/1051C12N9/1085C12N9/1205C12N9/90C12P7/22C12P19/44C12Y205/01054C12Y207/01071C12Y504/99005
Inventor 刘涛江晶洁殷华庄以彬马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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