A kind of genetically engineered bacteria producing l-histidine and its application

A technology of genetically engineered bacteria and histidine, applied in the field of genetic engineering

Active Publication Date: 2021-07-06
浙江震元生物科技有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • A kind of genetically engineered bacteria producing l-histidine and its application
  • A kind of genetically engineered bacteria producing l-histidine and its application
  • A kind of genetically engineered bacteria producing l-histidine and its application

Examples

Experimental program
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Embodiment 1

[0034] Construction of strain E. coli WHY3-1.

[0035] 1 Methods of gene editing

[0036] 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 The two plasmid maps used are in the appendix figure 1 . 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.

[0037] The specific steps of this method are as follows:

[0038] 1.1 pGRB plasmid construction

[0039] The purpose of constructing the plas...

Embodiment 2

[0101] Utilize described genetically engineered bacteria E.coli WHY3-1 to ferment and produce histidine as follows:

[0102] (1) Cultured in shake flasks

[0103] Slant culture: Streak inoculation of -80°C preserved strains on the activated slant, culture at 37°C for 12 hours, and passage once;

[0104] 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 6-8h;

[0105]Shake flask fermentation culture: Inoculate 10-15% inoculum into a 500mL Erlenmeyer flask with fermentation medium (final volume is 30mL), seal with nine layers of gauze, 37°C, 200r / min shaking culture, during the fermentation process by supplementing Add ammonia to maintain pH at 7.0-7.2; add 60% (m / v) glucose solution to maintain fermentation;

[0106] The composition of the slant medium is: glucose 1-5g / L, peptone 5-10g / L, beef extract 5-1...

Embodiment 3

[0117] Fermentation experiment of E.coli WHY3-1 on 5L tank.

[0118] The strain E.coli WHY3-1 constructed in Example 1 is used as a production strain to produce histidine:

[0119] Slant surface activation: Streak inoculate the preserved strains of glycerol on the slant medium of the test tube, and incubate at 37°C for 12;

[0120] Seed culture: Take one activated fresh eggplant-shaped bottle slope, 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 until the OD600 is about 15;

[0121] Fermentation tank culture: The fermenter seeds are connected to the seed solution with 15% inoculation amount (feeding to 450mL, and poured into the sterilized fermentation medium under fl...

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Abstract

The present invention provides a kind of genetically engineered bacterium producing L-histidine and application thereof, and this bacterium integrates nucleotide sequence such as Corynebacterium glutamicum ATP as shown in SEQ ID NO:1 on the genome of Escherichia coli Phosphoribosylase HisG Mutant Encoding Gene hisG * And make it strongly expressed to enhance the activity of HisG, the key enzyme of histidine synthesis; also increased the copy number of Escherichia coli's own histidine operon gene hisDBCHAFI on the genome, thereby enhancing the terminal synthesis pathway of histidine; The gene lysE encoding the arginine/lysine transporter derived from Corynebacterium glutamicum was integrated into the genome and strongly expressed to promote the secretion of intracellular histidine to the outside; also integrated into the genome The glutamate dehydrogenase encoding gene rocG of Bacillus was strongly expressed to promote the production of histidine.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a genetically engineered bacterium producing L-histidine and its construction method and application. Background technique [0002] L-histidine (hereinafter referred to as "histidine") is an important functional amino acid that participates in various physiological and biochemical processes such as body development, anti-oxidation and immune regulation. It is mainly used in the food, feed and pharmaceutical industries. It can be used as a nutritional fortifier and feed additive, as well as in the production of amino acid infusion and comprehensive amino acid preparations, to assist in the treatment of diseases such as heart disease, anemia and gastrointestinal ulcers. It has high economic and social value. [0003] At present, histidine is mainly produced by extracting from protein hydrolyzate, but this method has high loss rate, serious equipment corrosi...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/90C12N15/53C12N15/54C12N15/31C12P13/24C12R1/19
CPCC07K14/245C07K14/34C12N9/0016C12N9/1077C12N15/70C12N15/902C12P13/24C12Y104/01002C12Y204/02017
Inventor 谢希贤樊伟明吴鹤云蒋卫田道光陈燕娜张悦屠建情
Owner 浙江震元生物科技有限公司
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