High-yield L-histidine genetically engineered bacterium strain, constructing method and application thereof

A technology of genetically engineered bacteria and histidine, applied in the field of genetic engineering, can solve problems such as low fermentation performance that cannot meet the requirements of industrial production

Inactive Publication Date: 2019-08-30
TIANJIN UNIV OF SCI & TECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

V.G.Doroshenko et al. took wild Escherichia coli MG1655+ as the starting strain, knocked out the histidine operon leader peptide gene hisL, the transcriptional regulator purR, and introduced the point mutation E271K in the key enzyme HisG of histidine synthesis, so that the strain has the composition Amino acid accumulation ability, shake flask fermentation can accumulate histidine 4.9g / L, but its fermentation performance is too low to meet the requirements of industrial production

Method used

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  • High-yield L-histidine genetically engineered bacterium strain, constructing method and application thereof
  • High-yield L-histidine genetically engineered bacterium strain, constructing method and application thereof
  • High-yield L-histidine genetically engineered bacterium strain, constructing method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0045] Construction of strain E.coli WHY3:

[0046] 1 Methods of gene editing

[0047] 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 edited genome editing. Metabolic engineering, 2015, 31:13-21.), the method used The two plasmid maps of the attached 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.

[0048] The concrete steps of this method are as follows:

[0049] 1.1 pGRB plasmid construction

[0050] The purpose of constructing the plasmid pGR...

Embodiment 2

[0109] The method for producing histidine by fermentation of genetically engineered bacteria E.coli WHY3 is as follows:

[0110] Slant culture: Streak inoculation of -80°C preserved strains on the activated slant, culture at 37°C for 12 hours, and passage once;

[0111] Shake flask seed culture: Scrape a ring of slanted seeds with an inoculation loop and inoculate in a 500mL Erlenmeyer flask containing 30mL seed medium, seal with nine layers of gauze, and incubate at 37°C and 200rpm for 6-8h;

[0112] 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 water to maintain the pH at 7.0-7.2; add 60% (m / v) glucose solution to maintain the fermentation; the fermentation period is 24-30h;

[0113] The composition of the slant medium is: glucose 1-5g / L, peptone 5-10g / L, beef...

Embodiment 3

[0126] Shake flask fermentation experiments with the bacterial strain E.coli WHY2-3 constructed in Example 1 and the bacterial strain E.coli WHY3:

[0127] The above-mentioned two strains adopt the method of shaking flask fermentation to produce L-histidine under the same conditions, specifically as follows:

[0128] Slant culture: Streak inoculation of -80°C preserved strains on the activated slant, culture at 37°C for 12 hours, and passage once;

[0129] 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 8h;

[0130] Shake flask fermentation culture: Inoculate 15% inoculum into a 500mL Erlenmeyer flask containing fermentation medium (final volume is 30mL), seal with nine layers of gauze, culture at 37°C, 200r / min shaking, add ammonia water during fermentation Maintain the pH at 7.0-7.2; add 60% (m / v) glu...

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Abstract

The present invention provides a high-yield L-histidine genetically engineered bacterium strain and a constructing method thereof. The bacterium integrates a nucleotide sequence of a corynebacterium glutamicum ATP phosphoribosyl transferase HisG mutant encoding gene hisG* represented by SEQ ID NO:1 based on genome of escherichia coli to enhance activity of histidine-synthesizing key enzyme HisG, also increases copy number of histidine operon genes in the genome to enhance a terminal synthesis pathway of histidine, and also integrates an encoding gene lysE of arginine / lysine transportprotein derived from the corynebacterium glutamicum to promote extracellular secretion of the intracellular histidine. The genetically engineered bacterium is used for producing the L-histidine by a fermentation method, can stably produce 40-55 g / L of the histidine by fermentation in a 5L fermentation tank for 40-50 h, has a production intensity reaching 1.0-1.5 g / (Lxh), and has a conversion rate of 0.18-0.22 g histidine / g glucose.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a genetically engineered bacterium with high L-histidine production and its construction method and application. Background technique [0002] L-histidine is a protein structural element and the ninth essential amino acid for humans and animals. It participates in various physiological and biochemical processes such as body development, anti-oxidation and immune regulation. In addition to being used as a nutritional enhancer and feed additive, L-histidine is also increasingly used in the pharmaceutical industry, especially for the production of amino acid infusions and comprehensive amino acid preparations for diseases such as heart disease, anemia, and gastrointestinal ulcers Treatment. Therefore, histidine is an important amino acid product for human production and life, and has high economic and social value. [0003] The current production methods of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P13/24C12R1/19
CPCC07K14/34C12N9/1077C12P13/24C12Y204/02017
Inventor 谢希贤张悦樊伟明蒋卫吴鹤云陈燕娜田道光屠建情
Owner TIANJIN UNIV OF SCI & TECH
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