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Engineering strain for fermented synthesis of ergothioneine and construction method thereof

An engineering strain and ergothioneine technology, applied in the field of bioengineering, can solve the problems of expensive synthetic raw materials, high extraction cost and high synthetic cost, and achieve the effects of efficient biosynthesis and wide application value

Active Publication Date: 2019-10-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is very difficult to synthesize levorotatory ergothioneine by chemical methods, the safety of the product is difficult to guarantee, the synthetic raw materials are expensive, the synthetic cost is high, and the expected output is not reached; there are also many difficulties in the production of ergothioneine by the extraction method, such as insufficient raw material sources, raw material The content of ergothioneine is still very low, the extraction cost is high, and there are drug residues. With the rapid development of biological sciences such as molecular biology and metabolic engineering, the production of ergothioneine by biofermentation synthesis has become the most potential production method
[0004] However, in existing reports, the effect of producing ergothioneine by constructing genetically engineered bacteria is not satisfactory.

Method used

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  • Engineering strain for fermented synthesis of ergothioneine and construction method thereof
  • Engineering strain for fermented synthesis of ergothioneine and construction method thereof
  • Engineering strain for fermented synthesis of ergothioneine and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 124

[0045] Example 1 Shake Flask Culture and Ergothioneine Content Determination of 24 Escherichia coli Recombinant Bacteria Strains Heterologously Expressing the egtABCDE Gene Cluster

[0046] Pick 24 Escherichia coli recombinant strains with heterologous expression of egtABCDE gene cluster constructed above and single clones of control bacteria (transformed pEBS empty plasmid) and inoculate them in 5 mL LB medium, and add kanamycin at a final concentration of 50 μg / mL as required , placed at 200rpm at 37°C for 10-12h, and then transferred to a 250mL Erlenmeyer flask with a 10% inoculum size. Kanamycin, then placed at 220rpm and cultured at 37°C, methionine, histidine betaine, histidine, and cysteine ​​were added when culturing for 10 hours, and the final concentration of added methionine was 2g / L. The final concentration of histidine betaine is 10g / L, the final concentration of added histidine is 15g / L, the final concentration of added cysteine ​​is 15g / L, and the final liquid v...

Embodiment 218

[0047] Example 2 Shake Flask Culture and Ergothioneine Content Determination of 18 Bacillus subtilis Recombinant Bacteria Strains Heterologously Expressing the egtABCDE Gene Cluster

[0048] Pick 18 recombinant strains of Bacillus subtilis with heterologous expression of the egtABCDE gene cluster constructed above and single clones of control bacteria (transformed with pEBS empty plasmid) and inoculate them in 5 mL LB medium, and add Kanamyces at a final concentration of 50 μg / mL as required cultured at 200rpm at 37°C for 10-12h, and then transferred to a 250mL Erlenmeyer flask with 10% inoculum size. Kanamycin, then cultured at 220rpm at 37°C, added methionine, histidine betaine, histidine, cysteine ​​when cultured for 10h, and the final concentration of added methionine was 2g / L , the final concentration of histidine betaine added is 10g / L, the final concentration of histidine added is 15g / L, the final concentration of cysteine ​​added is 15g / L, and the final liquid volume i...

Embodiment 3

[0049] Example 3 Shake flask culture and ergothioneine content determination of Escherichia coli and Bacillus subtilis recombinant bacteria expressed in different combinations of genes contained in the egtABCDE gene cluster

[0050] Select Escherichia coli, Bacillus subtilis recombinant bacteria and control bacteria (transformed with pEBS empty plasmid) with optimized expression of different combinations of genes contained in the egtABCDE gene cluster constructed above and inoculate them in 5 mL LB medium, and add a final concentration of 50 μg as needed / mL kanamycin, cultured at 200rpm 37°C for 10-12h, and then transferred to a 250mL Erlenmeyer shaker flask with 10% inoculum size. Kanamycin with a final concentration of 50 μg / mL was then placed at 220 rpm and cultured at 37°C. Added methionine, histidine betaine, histidine, cysteine, and added methylthio The final concentration of amino acid is 8g / L, the final concentration of added histidine betaine is 15g / L, the final conc...

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Abstract

The invention discloses an engineering strain for fermented synthesis of ergothioneine and a construction method thereof, and belongs to the technical field of bioengineering. With escherichia coli and bacillus subtilis as the host and three shuttle plasmids pEBS of bacillus subtilis, escherichia coli and saccharomyces cerevisiae as the expression vector, egtABCDE gene clusters from different sources are introduced into the escherichia coli and the bacillus subtilis for heterologous expression; biosynthesis of the ergothioneine is realized; then, the egtABCDE gene clusters are subjected to optimized expression, including codon optimization, promoter optimization and expression of different combinations, and meanwhile a culture medium and added methionine, hercynine, histidine and cysteineare optimized. Through the optimized expression and optimized culture conditions, efficient biosynthesis of the ergothione is realized. The engineering strain lays a foundation for efficient fermentedpreparation of ergothione by a microbial system and is suitable for industrial production and application.

Description

technical field [0001] The invention relates to an engineering strain for fermenting and synthesizing ergothioneine and a construction method thereof, belonging to the technical field of bioengineering. Background technique [0002] Ergothioneine is a rare natural chiral histidine-derived thiol compound with unique biological functions and pharmacological activities. It not only has strong antioxidant activity: it can scavenge free radicals, peroxynitroso, hypochlorous acid, chelate divalent metal ions, activate antioxidant enzymes, inhibit superoxide dismutase, etc., but also It has anti-inflammatory and cell-protecting effects, and has broad application prospects and market prospects in the fields of medicine, food, health care products, cosmetics and biotechnology. [0003] Ergothioneine was first isolated from ergot bacteria in 1909, and it can also be synthesized in microorganisms such as actinomycetes, cyanobacteria and fission yeast, some fungi, streptomyces, mycobac...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12P13/04C12R1/125C12R1/19
CPCC12N9/00C12N15/75C12P13/04
Inventor 康振陈坚堵国成韦朝宝王丽
Owner JIANGNAN UNIV
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