Genetically engineered strain for producing ergothioneine and application

A technology of genetically engineered bacteria and ergothioneine, applied in the field of genetic engineering, can solve problems such as low yield and production intensity

Active Publication Date: 2021-01-22
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Under the condition of supplementing various precursors, Escherichia coli achieved t

Method used

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  • Genetically engineered strain for producing ergothioneine and application
  • Genetically engineered strain for producing ergothioneine and application
  • Genetically engineered strain for producing ergothioneine and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Construction of genetically engineered bacteria producing ergothioneine.

[0049] 1 Methods of gene editing

[0050] The gene editing method used in the present invention is carried out with reference to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR–Cas9 mediated genome editing. Metabolic engineering, 2015, 31:13-21.), the method See Figure 1 for the two plasmid maps used. Among them, pREDCas9 carries gRNA expression plasmid pGRB elimination system, bacteriophage λ Red recombination system and Cas9 protein expression system, spectinomycin resistance (working concentration: 100mg / L), cultured at 32°C; pGRB uses pUC18 as the backbone, including the promoter J23100, gRNA-Cas9 binding region sequence and terminator sequence, ampicillin resistance (working concentration: 100mg / L), cultured at 37°C.

[0051] The concrete steps of this method are as follows:

[0052] 1.1 pGRB plasmid construction

[0053] The purpose of...

Embodiment 2

[0128] Utilize the method for the production of ergothioneine by shake flask fermentation of the genetically engineered bacteria E.coli EGT5 and E.coli EGT6, E.coli EGT9, E.coli EGT10, E.coli EGT11 and E.coli EGT12 constructed in Example 1 as follows :

[0129] Slant culture: Streak inoculation of -80°C preserved strains on the activated slant, culture at 37°C for 12 hours, and passage once;

[0130] Shake flask seed culture: Scrape a ring of slanted seeds with an inoculation loop and inoculate in a 500mL Erlenmeyer flask containing 30mL of seed medium, seal with nine layers of gauze, and incubate at 37°C and 200rpm for 8-10h;

[0131] Shake flask fermentation culture: Inoculate the seed liquid into a 500mL Erlenmeyer flask with fermentation medium (final volume is 30mL) according to the inoculation amount of 10-15%, seal with nine layers of gauze, 37°C, 200r / min shaking culture, during the fermentation process Maintain the pH at 7.0-7.2 by adding ammonia water; add 60% (m / v)...

Embodiment 3

[0141] Fermentation experiment of E.coli EGT12 in 5L tank.

[0142] The bacterial strain E.coli EGT12 that embodiment 1 builds produces ergothioneine as production bacterial strain, specifically as follows:

[0143] Slant activation: Streak inoculate the preserved strains of glycerol on the slant medium of the test tube and incubate at 37°C for 12 hours; then streak the preserved strains on the slant medium of the eggplant-shaped bottle and incubate at 37°C for 14 hours. Seed culture: Take a slant of an activated fresh eggplant-shaped bottle, wash it with 150mL sterile water, inoculate it into a fermenter under flame protection, control the temperature at 37°C, automatically add ammonia water to control the pH at 7.0, and the initial aeration rate is 2L / min, the initial stirring speed is 200rpm, the DO value is maintained between 20-30% during the cultivation process, and the seeds are cultivated to OD 600 It is around 15.

[0144] Fermentation tank culture: the seeds of th...

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Abstract

The invention provides a genetically engineered strain for high yield of ergothioneine and application. The stain takes escherichia coli as a host, and a coding gene hisG* of a corynebacterium glutamicum ATP phosphoribosyl transferase HisG mutant is integrated on a genome of the host; the copy number of a histidine operon gene hisDBCHAFI is further increased on the genome of the host; a mycobacterium smegmatis ergothioneine operon gene egtBCDE is further integrated on the genome of the host; an Escherichia coli glutamylcysteine ligase encoding gene gshA is integrated on the genome of the host,so that the synthesis of the ergothioneine is promoted; a neurospora crassa C-S lyase coding gene egtE<ncr> is further integrated on the genome of the host, so that the synthesis of ergothioneine isfurther promoted; and a gene egtB*<msm> of a sulfoxide synthase mutant is integrated on a genome of the host, so that synthesis of ergothioneine is further promoted.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a genetically engineered bacterium for producing ergothioneine and its application. Background technique [0002] Ergothioneine (EGT), mercaptohistidine trimethyl inner salt, is a natural amino acid derivative discovered in ergot in 1909. Ergothioneine has multiple functions such as anti-oxidation, scavenging free radicals, regulating redox reactions, and participating in intracellular energy regulation. Due to its excellent cell physiological protection function, ergothioneine can be applied to many fields including medicine, food, feed and cosmetic industries. In 2017, ergothioneine was approved by the European Food Safety Authority as a safe new resource food for infants, pregnant women and lactating women. In 2019, Ergothioneine was certified as GRAS by the US Food and Drug Administration. [0003] At present, the main production method of ergothioneine is subm...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/60C12N15/54C12N15/52C12N15/90C12P13/24C12R1/19
CPCC12N15/52C12N9/1077C12N9/93C12N9/88C12N15/70C12N15/902C12P13/24C12Y204/02017C12Y603/02002
Inventor 马倩田道光谢希贤吴鹤云李镠蒋帅秦臻朱彦凯王加初朱永铎
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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