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Genetically engineered bacterium for producing hydroxyectoine and application of genetically engineered bacterium

A technology of hydroxytetrahydropyrimidine and genetic engineering bacteria, applied in the field of genetic engineering, can solve the problems of long production cycle, high market price, low yield, etc., and achieve the effect of high-efficiency synthesis

Active Publication Date: 2021-06-01
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The process requires high equipment and high energy consumption. In addition, the high-salt environment is not conducive to the growth of bacteria, resulting in a long production cycle and low synthesis efficiency of the target product. The product is usually a mixture of hydroxy ectoine and ectoine, which requires further use. Chromatographic separation technology for product purification
Therefore, the yield of hydroxy ectoine is low, the cost is high, and the market price is very expensive
The problems of high by-product content, high cost, and low yield have seriously restricted the industrial production and application of hydroxyectoine, and there is an urgent need to construct a genetically engineered strain that efficiently synthesizes a single product

Method used

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  • Genetically engineered bacterium for producing hydroxyectoine and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing hydroxyectoine and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing hydroxyectoine and application of genetically engineered bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Construction of E.coli W3110 Hydroxytetrahydropyrimidine Genetically Engineered Bacteria

[0048] Targeted gene modification using CRISPR / Cas9 gene editing technology. The gene editing method adopted in the present invention is carried out with reference to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichiacoli using CRISPR-Cas9 meditated genome editing. Metabolic engineering, 2015, 31:13-21.).

[0049] The specific steps of this method are as follows:

[0050] (1) Construction of pGRB plasmid:

[0051] Use CRISPR RGEN Tools to design a specific target sequence ((PAM: 5'-NGG-3') for cutting the target gene. After synthesizing forward primer and reverse complementary primer, take 10 μL of each into a PCR tube, and mix them evenly. A double-stranded fragment was formed by annealing single-stranded DNA by PCR. Reaction conditions: pre-denaturation at 95° C., 5 min; The pGRB plasmid was obtained. The kit used for homologous recom...

Embodiment 2

[0077] Example 2: The method for producing hydroxytetrahydropyrimidine by using the hydroxytetrahydropyrimidine genetically engineered bacteria obtained in step (2) in Example 1 to knock out the yeeL gene and integrate icd by shaking flask fermentation

[0078] (1) Activated slant culture: use an inoculation loop to inoculate 1-2 loops of bacteria from the -80°C refrigerator storage tube, spread evenly on the slant medium, cultivate at 37°C for 12 hours, and transfer to the second-generation slant medium , cultured at 37°C for 12h;

[0079] (2) Seed bottle culture: use an inoculation loop to inoculate the bacterial cells on the inclined surface into a 500mL conical flask containing 30 mL of seed medium for the preparation of seed liquid. Shake culture for 12h under the condition of min;

[0080] (3) Fermentation culture: the seed liquid was inoculated into a 500mL baffle bottle containing a fermentation medium according to 10% of the inoculum, so that the final volume was 30mL,...

Embodiment 3

[0085] Embodiment 3: utilize esaI / esaR quorum sensing circuit engineering bacteria shake flask fermentation method to produce hydroxytetrahydropyrimidine

[0086] (1) Activated slant culture: use an inoculation loop to inoculate 1-2 loops of bacteria from the -80°C refrigerator storage tube, spread evenly on the slant medium, cultivate at 37°C for 12 hours, and transfer to the second-generation slant medium , cultured at 37°C for 12h;

[0087] (2) Seed bottle culture: use an inoculation loop to inoculate the bacterial cells on the inclined surface into a 500mL conical flask containing 30 mL of seed medium for the preparation of seed liquid. Shake culture for 12h under the condition of min;

[0088] (3) Fermentation culture: the seed liquid was inoculated into a 500mL baffle bottle containing a fermentation medium according to 10% of the inoculum, so that the final volume was 30mL, sealed with twelve layers of gauze, at 37°C, 180r / min Under the condition of shaking culture, t...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to the construction and the application of a gene engineering bacterium for producing hydroxyectoine. According to the invention, key genes in a hydroxyectoine synthesis route are strengthened on the genome, a hydroxyectoine competitive inhibition route is weakened, the hydroxyectoine synthesis route is constructed, and an isocitrate dehydrogenase coding gene icd is over-expressed to increase the supply of a precursor alpha-ketoglutaric acid. Meanwhile, supply of a precursor substance alpha-ketoglutaric acid is dynamically adjusted and controlled through an esaI / esaR quorum sensing circuit strategy, metabolism of the alpha-ketoglutaric acid in tricarboxylic acid circulation is automatically adjusted according to the cell density, and the hydroxyectoine high-yield strain is obtained. The constructed strain can realize efficient synthesis of hydroxyectoine under the fermentation condition that alpha-ketoglutaric acid is not added, does not accumulate a byproduct ectoine, and has important industrial application value.

Description

Technical field: [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the construction and application of a genetically engineered bacterium for producing hydroxytetrahydropyrimidine. Background technique: [0002] Hydroxytetrahydropyrimidine ((4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) is a hydroxylated derivative of tetrahydropyrimidine and is considered to be the most widely distributed one of the compatible solutes. It is distinguished from other compatible solutes due to its special biological properties, such as its suitability for improving the thermotolerance of proteins and cells, making it a target of economic interest. [0003] Hydroxytetrahydropyrimidine is used to stabilize macromolecules and whole cells, stabilize enzymatic activity in vitro, promote protein folding in vivo, protect molecules and cells against freeze-thaw cycles, promote protection against desiccation, oxidation...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/54C12N15/53C12N15/60C12N15/113C12N15/31C12P17/12C12R1/19
CPCC12N9/88C12N9/1247C12N9/0006C12N9/0008C12N9/1029C12N9/1217C12N9/0071C07K14/195C12P17/12C12Y402/01108C12Y207/07006C12Y401/01031C12Y101/01041C12Y102/01052C12Y203/01184C12Y207/02004C12Y114/11
Inventor 马倩夏利谢希贤陈宁谭淼孙全伟张颖杨蒙雅徐庆阳张成林李燕军范晓光
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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