Method for constructing L-ergothionine producing strain

An ergothioneine and gene expression technology, applied in the field of genetic engineering, can solve problems such as concerns about application prospects, and achieve good industrial application potential and stable genetic traits

Active Publication Date: 2020-08-14
ZHEJIANG HUARUI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as an opportunistic pathogen and a genetically modified strain, its product application prospects are worrying

Method used

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  • Method for constructing L-ergothionine producing strain
  • Method for constructing L-ergothionine producing strain
  • Method for constructing L-ergothionine producing strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1: original strain screening

[0064] According to the retrieved literature reports, the food-source safe microorganisms capable of producing ergothioneine were screened. Several strains of Rhodotorula torula were purchased from the China General Microorganism Collection Center for preliminary screening and comparison, and the ergothioneine production of each strain was evaluated.

[0065] Inoculate the purchased strains in the yeast medium for shake-flask fermentation. Yeast seed medium composition: glucose 20g / L, yeast powder 10g / L, peptone 20g / L.

[0066] Glycerol tube seeds are inoculated with seed medium, cultured at 30°C, 250 rpm, for 17 hours, and can be transferred when OD600>16.

[0067] Yeast shake flask fermentation medium: ammonium sulfate 0.5g / L, yeast powder 10g / L, peptone 10g / L, glutamic acid 3g / L, biotin 0.6mg / L, thiamine hydrochloride 0.1g / L, magnesium sulfate 5g / L, ferrous sulfate heptahydrate 0.278g / L, manganese sulfate monohydrate 0.085...

Embodiment 2

[0073] Example 2: Construction of plasmid pZPK-PGPD-Hyg-Tnos

[0074] The genetic manipulation method of Rhodotorula torutorula can refer to the following literature: Lin, X., et al. (2014). "Functional integration of multiple genes into the genome of the oleaginous yeast Rhodosporidium toruloides." FEMS Yeast Research 14(4):547- 555.

[0075] Referring to this document, according to the published sequence, determine the complete sequence of the plasmid pZPK-PGPD-Hyg-Tnos, and send it to Suzhou Jinweizhi Biotechnology Co., Ltd. for synthesis to obtain the plasmid pZPK-PGPD-Hyg-Tnos (SH205), whose structure is as follows figure 1 Shown, its base sequence is SEQ ID NO: 4, 8378bp.

[0076] Use this as the plasmid backbone for subsequent plasmid construction.

Embodiment 3

[0077] Embodiment 3: Construction EGT1 gene expression cassette and expression plasmid

[0078] For the target genes NcEGT1, CpEGT1 and RmEGT1 derived from three microorganisms, pGPD was designed as a promoter to control the expression of the functional gene; the CYC1t terminator derived from Saccharomyces cerevisiae was used to control the expression termination of the functional gene; and at the beginning and end Design and add XbaI restriction site (tctaga) and PmeI restriction site (gtttaaac) respectively, so as to be used for subsequent plasmid construction. The designed gene expression cassettes are respectively Pgpd-NcEGT1-ScCYC1t (ie SEQ ID NO:1), Pgpd-CpEGT1-ScCYC1t (ie SEQ ID NO:2) and Pgpd-RmEGT1-ScCYC1t (ie SEQ ID NO:3)

[0079] The sequence of the designed gene expression cassette was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for synthesis, and the above gene expression cassette was cloned on the Puc57 plasmid to obtain the EGT1 gene expression plasmid, whi...

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Abstract

The invention discloses a rhodotorula glutinis engineering bacterium and a construction method thereof. The rhodotorula glutinis engineering bacterium can express exogenous egt1 enzyme so as to improve the capacity of producing L-ergothionine, the yield of the fermented L-ergothionine is close to 1.5 g / L, and the engineering bacterium is stable in inheritable character and has an industrial application prospect.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a method for constructing ergothioneine-producing bacteria, in particular to a method for constructing Rhodotorula torula for producing ergothioneine. Background technique [0002] Ergothioneine (L-ergothionine, EGT), the chemical name is 2-mercaptohistidine trimethyl inner salt, and its structural formula is as follows: [0003] [0004] Ergothioneine is the only known natural 2-thioimidazole amino acid. Ergothioneine has various physiological effects such as anti-oxidation, anti-inflammation, prolonging cell survival cycle or anti-aging activity, and improving nerve cell generation. At the same time, it has a good effect of protecting cells and resisting damage in various disease models, including Alzheimer's, diabetes and other complications, and has a broad market application prospect. [0005] At present, ergothioneine can be obtained through chemical synthesis, edible ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/66C12N15/53C12N1/19C12P13/04C12R1/645
CPCC12N15/815C12N15/66C12N9/0083C12P13/04C12Y114/99C12N2800/22
Inventor 范文超高书良王金刚任亮俞想
Owner ZHEJIANG HUARUI BIOTECHNOLOGY CO LTD
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