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Recombinant saccharomyces cerevisiae strain and fermentation method thereof

A technology of combining enzymes and ergot alkaloids, applied in the field of synthetic biology, can solve problems such as obstruction, failure to achieve full synthesis, difficulty in EasE protein expression, etc.

Pending Publication Date: 2021-07-27
TIANJIN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2014, researchers such as CurtAF Nielsen successfully synthesized basalline in Saccharomyces cerevisiae with tryptophan and DMAPP, and screened the two proteins EasE and EasC from Aspergillus japonica to realize the synthesis of basalline, making EasE and It is possible to express EasC in heterologous yeast. The study of the key enzymes EasE and EasC relies on the gene complementation of fungi, but further characterization is hindered by the difficulty of expressing EasE protein
[0005] As an intermediate product towards an important branch point in the synthetic pathway of ergot alkaloids, ergot alkaloid plays a pivotal role in the development of biosynthetic lysergic acid derivatives series, and among ergot alkaloids, lysergic acid derivatives are mainly used in pharmaceutical development One type has not been fully synthesized in Saccharomyces cerevisiae so far, so the successful heterologous expression of ergot alkaloids is of great significance to the research of downstream ergot alkaloids

Method used

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  • Recombinant saccharomyces cerevisiae strain and fermentation method thereof
  • Recombinant saccharomyces cerevisiae strain and fermentation method thereof
  • Recombinant saccharomyces cerevisiae strain and fermentation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0059] Example 1 The acquisition of recombinant brewer's yeast producing ergot alkaloids

[0060] Provided by Yuan Yingjin's research group, the strain number is SyBE_Sc06130032. The genetic modification contained in the recombinant Saccharomyces cerevisiae is: insert dmaW-easF at the Δ22 site of the XV chromosome, insert easE-easC into the Δ15 site of the XVI chromosome, insert ero1-fad1 into the Δ14 site of the VIII chromosome, and insert easD is inserted into the GAL1 / 7 / 10 position, and easG-Pgal110-easA is connected into the wn416 functional cassette, which exists in the form of a plasmid.

[0061] The specific steps are:

[0062] Obtaining High-yielding DMAPP Recombinant Saccharomyces cerevisiae (Chassis Strain)

[0063] The chassis strain was provided by Yuan Yingjin's research group at Tianjin University, and the strain number is yCTH. The genetic modification contained in the recombinant Saccharomyces cerevisiae is as follows: at GAL80, IDI1 and tHMGR are overexpres...

Embodiment 2

[0110] Example 2 Construction of Cofactor Regulated Modular Integration Plasmid

[0111] Considering the existence of the EasG-EasA plasmid module containing the uracil nutritional tag in the chassis strain, the authors designed to insert the cofactor regulatory enzymes UTR1, MAE1, POS5, and PYC2 into the functional expression cassette WN414, and digested the functional expression cassette WN414 with BsaI enzyme, in A nick is obtained in the middle of Pgal1-TDH1t to obtain a linearized vector, and the homology arms of each 20bp at the two nicks of the vector are added to the two ends of UTR1, MAE1, POS5, and PYC2 by means of a PCR instrument through upper and lower primers, and used in vitro The seamless cloning method was used for connection, and the connection system was introduced into DH5α together, and the transformants were selected for amplification and culture to extract plasmids for sequencing. The sequenced plasmids were transformed into SyBE_Sc06130032 by the lithium...

Embodiment 3

[0116] Experimental bacteria fermentation detection of embodiment 3 cofactor regulation

[0117] Test material: strain SyBE_Sc06130102-SyBE_Sc06130106

[0118] experiment method:

[0119] Seed medium: SD-URA liquid medium (synthetic yeast nitrogen source YNB 6.7g / L, glucose 20g / L, mixed amino acid powder lacking tryptophan, leucine, histidine and uracil 2g / L, color amino acid, leucine, histidine in the form of 100* mother solution);

[0120] Fermentation medium: SD-URA liquid medium (synthetic yeast nitrogen source YNB 6.7g / L, glucose 40g / L, mixed amino acid powder 2g / L lacking tryptophan, leucine, histidine and uracil, color amino acid, leucine, histidine in the form of 100* mother solution).

[0121] Inoculate the above strains into 5mL seed medium, culture at 30°C and 250rpm for 14-16h, and use the initial cell concentration OD 600 =0.2 were inoculated in 50 mL of fermentation medium, cultivated for 120 h at 22° C. and 220 rpm, and monitored the bacterium density (OD600...

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Abstract

The invention relates to the field of synthetic biology, in particular to a recombinant Saccharomyces cerevisiae strain and a fermentation method of the recombinant Saccharomyces cerevisiae strain. According to the invention, the necessity of a precursor substrate and a methyl donor to a whole heterologous path is determined, and the heterologous synthesis path of agroclavine generates cofactor imbalance disturbance to endogenous metabolism, so that favorable evidence is provided for subsequent continuous optimization and yield increase of agroclavine. The yield of the recombinant saccharomyces cerevisiae strain disclosed by the invention is obviously improved compared with that of the currently reported strain.

Description

technical field [0001] The invention relates to the field of synthetic biology, in particular to a recombinant Saccharomyces cerevisiae strain and a fermentation method thereof. Background technique [0002] Ergot alkaloids are mainly secondary metabolites of Aspergillus, Claviceps, and Neotyphodium, which are fungal indole-like organisms with strong pharmacological activity. Alkaline natural products, raw materials for therapeutic drugs for migraines, uterine bleeding, Parkinson's disease and other diseases. Alkaloid drugs are often semi-synthesized from plant-derived starting materials, which often limits their availability and final price. Recent advances in synthetic biology have made it possible to introduce complete plant pathways into microorganisms to produce plant alkaloids. [0003] The production of modified alkaloids by microorganisms has the potential to accelerate the semi-synthesis of alkaloid drugs, which provides advanced intermediates that are structurall...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/54C12N1/19C12P17/18C12R1/865
CPCC12N15/81C12N9/1205C12P17/182C12Y207/0104C12Y207/01086
Inventor 元英进吴楠杜现礼王颖姚明东段小涛肖文海
Owner TIANJIN UNIV
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