Genetically engineered bacterium for producing N-acetylneuraminic acid as well as construction and application of genetically engineered bacterium

A technology of genetically engineered bacteria and genes, applied in the field of genetic engineering, can solve problems such as inability to meet the needs of industrial production

Active Publication Date: 2021-12-21
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above yields are the highest yields in different host cells reported so far, but still cannot meet the needs of industrial production

Method used

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  • Genetically engineered bacterium for producing N-acetylneuraminic acid as well as construction and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing N-acetylneuraminic acid as well as construction and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing N-acetylneuraminic acid as well as construction and application of genetically engineered bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1: Neu5Ac produces the construction of Escherichia coli genetically engineered bacteria

[0052] Directed gene modification using CRISPR / Cas9 gene editing technology. 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 Escherichiacoli using CRISPR–Cas9 mediated genome editing. Metabolic engineering, 2015, 31:13-21.).

[0053] Some of the experimental methods involved are as follows:

[0054] (1) Construction of pGRB plasmid:

[0055] The target sequence (PAM:5'-NGG-3') was designed using CRISPR RGEN Tools for cleavage of the target gene. After synthesizing the forward primer and the reverse complementary primer, take 10 μL of each in a PCR tube, mix them evenly, and prepare a DNA fragment containing the target sequence by annealing single-stranded DNA. Reaction conditions: pre-denaturation at 95°C, 5min; annealing at 50°C, 1min. The obtained DNA fragme...

Embodiment 2

[0096] Example 2: A method for producing Neu5Ac using the strain constructed in Example 1 above as a production strain

[0097] (1) Slant surface activation culture: Take out the microbial strains stored at -80°C, inoculate them densely into the slant surface activation medium under sterile conditions, place them at 37°C for about 12 hours, and transfer them to the slant eggplant shape after the cultivation is completed. The bottle activation medium was subcultured once;

[0098] (2) Seed tank culture: Pour about 200mL of sterile water into an eggplant-shaped bottle near the flame of the ultra-clean bench, use an inoculation loop to scrape the colony into sterile water to prepare a bacterial suspension, and inoculate the bacterial suspension into the seed medium During the cultivation process, the pH was maintained at 7.0, the temperature was maintained at 37°C, and the dissolved oxygen was maintained at 30%, and the culture was carried out until the dry cell weight was 3g / L, ...

Embodiment 3

[0106] Example 3: A method for producing Neu5Ac using the strain constructed in Example 1 above as a production strain

[0107] (1) Slant surface activation culture: Take out the microbial strains stored at -80°C, inoculate them densely into the slant surface activation medium under sterile conditions, place them at 36°C for about 13 hours, and transfer them to the slant eggplant shape after the cultivation is completed. The bottle activation medium was subcultured once;

[0108] (2) Seed tank culture: Pour about 200mL of sterile water into an eggplant-shaped bottle near the flame of the ultra-clean bench, use an inoculation loop to scrape the colony into sterile water to prepare a bacterial suspension, and inoculate the bacterial suspension into the seed medium During the cultivation process, the pH was maintained at 7.2, the temperature was maintained at 36°C, and the dissolved oxygen was maintained at 25%, and the culture was carried out until the dry cell weight was 4g / L, ...

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Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a genetically engineered bacterium for producing N-acetylneuraminic acid through xylose induction. Escherichia coli is used as a starting strain, an N-acetylglucosamine synthesis pathway is integrated on a genome, an N-acetylglucosamine 2-epimerase gene bAGE and an N-acetylneuraminic acid synthetase gene neuB from collar algae are introduced, an N-acetylneuraminic acid synthesis pathway is constructed, and a key gene nano ATEK of a catabolism pathway of the N-acetylneuraminic acid is knocked out. Meanwhile, metabolic pathways of precursor substances required by synthesis of the N-acetylneuraminic acid are subjected to multi-copy reinforcement, part of bypass metabolic pathways are knocked out, key enzyme genes for producing GlcNAc and Neu5Ac are optimized according to different copy numbers, the optimal proportion of the key enzyme genes is finally determined, and the high-yield strain of the N-acetylneuraminic acid is obtained. The highest yield of the N-acetylneuraminic acid can reach 28g / L, the highest production intensity can reach 0.67 g / (L*h) which is the highest value reported at present, and the N-acetylneuraminic acid has important industrial application value.

Description

Technical field: [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a genetically engineered bacterium which utilizes xylose to induce the production of N-acetylneuraminic acid and its construction and application. Background technique: [0002] Neuraminic acid, also known as sialic acid, is an acidic amino sugar with nine carbon atoms and a pyranose structure. N-acetylneuraminic acid (Neu5Ac) is the most important member of the sialic acid family, which is of great significance in the process of life activities. [0003] N-acetylneuraminic acid (Neu5Ac) is commonly found at the non-reducing ends of glycolipids and glycoproteins and serves as a ligand receptor and cell-cell interactions during fertilization, immunity, differentiation and neural events. N-acetylneuraminic acid has many important functions, including improving learning and memory, promoting bone growth in babies, absorbing vitamins and minerals, and promot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/54C12N15/60C12N15/61C12N15/31C12P19/26C12R1/19
CPCC12N9/1247C12N9/1029C12N9/1096C12N9/1205C12N9/90C12N9/88C12N9/1085C12N1/20C07K14/245C07K14/195C12P19/26C12Y207/07006C12Y203/01004C12Y206/01016C12Y207/01C12Y501/03008C12Y205/01056C12Y207/01002C12Y207/0104C12Y402/03012Y02A50/30
Inventor 马倩张颖侯正杰谢希贤杨蒙雅卓明洋赵可欣陈宁徐庆阳张成林李燕军范晓光
Owner TIANJIN UNIV OF SCI & TECH
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