Engineering strain for xylose-induced production of N-acetylneuraminic acid and application of engineering strain

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

Active Publication Date: 2021-03-26
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The above yields are the highest yields in different host cells repo

Method used

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  • Engineering strain for xylose-induced production of N-acetylneuraminic acid and application of engineering strain
  • Engineering strain for xylose-induced production of N-acetylneuraminic acid and application of engineering strain
  • Engineering strain for xylose-induced production of N-acetylneuraminic acid and application of engineering strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: Construction of E.coli W3110 Neu5Ac genetically engineered bacteria

[0040] 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.).

[0041] The concrete steps of this method are as follows:

[0042] (1) Construction of pGRB plasmid:

[0043] 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 resulting DNA fragment was ligated with the ...

Embodiment 2

[0069] Embodiment 2: The method of producing Neu5Ac by fermentation of genetically engineered bacteria E.coli W3110 Neu5Ac

[0070] (1) Activated slant culture:

[0071] The composition of the slant medium is: yeast powder 5g / L, peptone 10g / L, NaCl 5g / L, beef extract 10g / L, sucrose 1g / L, agar powder 20g / L.

[0072] Use an inoculation loop to inoculate 2 rings of strains from a -80°C refrigerator bacteria-preserving tube, spread evenly on the slant medium, incubate at 37°C for 12 hours, transfer to the second-generation slant medium, and incubate at 37°C for 12 hours.

[0073] (2) Seed bottle culture:

[0074] The composition of the seed medium is: glucose 20g / L, yeast powder 3g / L, (NH 4 ) 2 SO 4 2g / L, KH 2 PO 4 2g / L, MgSO 4 ·7H 2 O 1g / L, citric acid 2g / L, FeSO 4 ·7H 2 O 2.8mg / L, MnSO 4 ·7H 2 O 1.2mg / L, V H 0.1mg / L, V B1 0.5mg / L, trace element mixture 1ml / L, 1 drop of defoamer, the rest is water, pH 7.0.

[0075] The composition of the trace element mixture is:...

Embodiment 3

[0081] Embodiment 3: Utilize the method for the fermentative production of Neu5Ac of genetically engineered bacteria E.coli W3110 Neu5Ac

[0082] (1) Activated slant culture: Use an inoculation loop to inoculate 1-2 loops of bacteria from a -80°C refrigerator bacteria-preserving tube, spread evenly on the slant medium, culture at 37°C for 12 hours, and transfer to the second-generation slant medium , cultivated at 37°C for 12h;

[0083] (2) Seed bottle cultivation: use an inoculation loop to inoculate the thalli on the inclined surface into a 500mL triangular flask equipped with 30mL seed culture medium to prepare the seed liquid. Under the condition of shaking for 12 hours;

[0084] (3) Fermentation culture: Inoculate the seed liquid into a 500mL baffle bottle equipped with a fermentation medium according to 10% inoculum amount, make the final volume 30mL, seal it with twelve layers of gauze, at 35°C, 180r / min Shaking culture under the conditions, maintaining the pH at 6.8-...

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Abstract

The invention belongs to the technical field of genetic engineering and particularly relates to construction and application of a genetic engineering strain for xylose-induced production of N-acetylneuraminic acid. According to the construction and the application, a N-acetylglucosamine synthesis way is integrated with a genome, a N-acetylglucosamine 2-epimerase gene bAGE and a N-acetylneuraminicacid lyase gene shNAL are introduced, a N-acetylneuraminic acid synthesis way is constructed, and a key gene nanTEK of a catabolism way of the N-acetylneuraminic acid synthesis way is knocked out. Meanwhile, metabolic ways of precursor substances required for synthesizing the N-acetylneuraminic acid are subjected to multicopy strengthening, and part of bypath metabolic ways are subjected to knockout, so that the N-acetylneuraminic acid high yielding strain is obtained. In case of shake-flask fermentation for 36h, the yield of the N-acetylneuraminic acid can reach 10.8g/L to the maximum, the production intensity can reach 0.3g/(L*h) which is the maximum value reported at present, and thus, the engineering strain has an 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 that utilizes xylose to induce the production of N-acetylneuraminic acid. 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. Neu5Ac is usually located at the end of glycoproteins and glycolipids on the cell membrane surface, and plays a key role in cell recognition and signal transmission. Reasonable intake of Neu5Ac can promote brain development of infants, maintain normal brain function of the elderly, and prevent Alzheimer's disease. Its derivative, zanamivir, has been designed to inhibit influenza A and B viruses, and some other de...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P19/26C12R1/19
CPCC12N15/52C12N9/1029C12N9/1247C12N9/88C12N9/90C12N9/80C12N9/78C12N9/1096C12N9/0008C12N9/0006C12N9/12C12N9/1217C12N9/1205C12N15/70C12P19/26C12Y101/01027C12Y207/07006C12Y203/01004C12Y206/01016C12Y501/03008C12Y401/03003C12Y305/01025C12Y305/99006C12Y102/05001C12Y207/02001C12Y203/01054
Inventor 马倩谭淼张颖杨蒙雅谢希贤陈宁徐庆阳李燕军张成林范晓光
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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