A kind of Saccharomyces cerevisiae recombinant strain and its construction method and application

A technology of Saccharomyces cerevisiae and its construction method, which is applied in the field of Saccharomyces cerevisiae recombinant bacteria and its construction, and can solve the problems of low catalytic efficiency of exogenous pathway enzymes, etc.

Active Publication Date: 2022-05-31
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Insufficient supply of precursors and low catalytic efficiency of exogenous pathway enzymes are the main reasons for limiting caffeic acid synthesis

Method used

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  • A kind of Saccharomyces cerevisiae recombinant strain and its construction method and application
  • A kind of Saccharomyces cerevisiae recombinant strain and its construction method and application
  • A kind of Saccharomyces cerevisiae recombinant strain and its construction method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

Bgl II was double digested, and the obtained fragment was the same as the pUMRI-13 double digested by EcoR I and Bgl II (GenBank:

KM216415.1) plasmid was connected for transformation, and the pUMRI-13-HpaC plasmid was constructed.

PCR reaction system is as follows:

[0065]

The PCR program is as follows:

[0067]

Plasmid or gene fragment restriction enzyme digestion system is as follows:

[0069]

[0070]

[0071] 37° C., digested with enzyme for 2h. The digested products were separated by 1.0% agarose gel electrophoresis, and the gel with gene fragments was excised.

And purified and recovered with a gel recovery kit.

[0073]

[0073]

22 DEG C, the enzyme is connected for 50min, and the ligation product is transformed into E. coli competent state, placed in a 37 DEG C incubator and cultivated

The positive clones obtained overnight were used for plasmid extraction.

Embodiment 2

[0080] Enzymatic cleavage conditions: place 50° C. for cleavage for 2-3 hours.

Embodiment 3

[0086] (2) Get 50 μL of supernatant into a new 1.5mL EP tube, add 950 μL of purified water and dilute 20 times.

[0087] (3) Use a 0.22 μm water-based needle filter to filter the obtained sample for the determination of caffeic acid.

[0088] Quantitative analysis of caffeic acid: the sample was analyzed using an Agilent 1200 high performance liquid chromatograph. Chromatographic conditions

[0089]

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Abstract

The invention discloses a Saccharomyces cerevisiae recombinant bacterium, which integrates in the genome RgTAL Gene, HpaB genes and HpaC Gene. The invention also discloses the construction method and application of Saccharomyces cerevisiae recombinant bacteria. Finally, the invention also discloses a production method of caffeic acid. The present invention integrates three exogenous genes required for caffeic acid synthesis into the chromosome of Saccharomyces cerevisiae, and through the knockout of competition pathway genes, the elimination of feedback inhibition steps, and the overexpression of rate-limiting enzymes, the transition from glucose to For the total synthesis of caffeic acid, the shake flask yield reached about 760 mg / L, which is the highest level of yeast yield reported so far, which provides a new method for the industrial production of caffeic acid.

Description

A kind of Saccharomyces cerevisiae recombinant bacteria and its construction method and application technical field The present invention belongs to the technical field of bioengineering, be specifically related to a kind of Saccharomyces cerevisiae recombinant bacteria and its construction method and application. use. Background technique Caffeic acid (Caffeic acid), is a kind of secondary metabolite widely distributed in plants, not only has anti- It has biological activities such as bacterial resistance and disease resistance, and is an important raw material and intermediate for medicine. The main source of caffeic acid is Chemical synthesis and plant extraction. The chemical synthesis of caffeic acid has many problems, such as many synthesis steps, complex process and unfriendly environment. With the development of synthetic biology technology, the use of microbial fermentation to produce caffeic acid has become a Important options. Existing patent (CN 108...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81C12P7/42C12R1/865
CPCC12N15/81C12N9/88C12N9/0071C12N9/001C12Y403/01025C12Y114/14009C12Y103/08C12P7/42Y02E50/10
Inventor 周萍萍岳春磊杜艺
Owner YANGZHOU UNIV
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