High-yield N-acetylneuraminic acid metabolic engineering bacterium and construction method and application

A technology of acetylneuraminic acid and metabolic engineering, which is applied in the field of high-yield N-acetylneuraminic acid metabolic engineering bacteria and construction, can solve the problems of restricting large-scale application, high cost, and low production of N-acetylneuraminic acid. Achieve the effect of improving synthesis efficiency and reducing the accumulation of by-products

Inactive Publication Date: 2015-10-21
武汉中科光谷绿色生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main problem of the fermentation method is that the production of N-acetylneuraminic acid in the current strain is too low, resulting in its high cost, which limits its large-scale application

Method used

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  • High-yield N-acetylneuraminic acid metabolic engineering bacterium and construction method and application
  • High-yield N-acetylneuraminic acid metabolic engineering bacterium and construction method and application
  • High-yield N-acetylneuraminic acid metabolic engineering bacterium and construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: Construction of high-yield N-acetylneuraminic acid genetically engineered bacteria

[0027] 1. Using the Red recombination method to knock out the nanATEK gene cluster in the strain, the specific steps are as follows:

[0028] 1. Knockout of nanATEK gene cluster

[0029] 1) According to the sequence of Escherichia coli BL21(DE3) genome (Genbank No.CP001509), design primers: upstream primer F-nanA:

[0030] GCAACGAATTTACGTGGCGTAATGGCTGCACTCCTGACTATTCCGGGGATCCGTCGACC (shown in SEQ ID NO.1) and downstream primer R-nanK:

[0031] TTTTTCTCCCCTGGGCCAACAGCGCAGCCCCAAGTAAACCTGTAGGCTGGAGCTGCTTC (shown in SEQ ID NO.2), using primers F-nanA and R-nanK, using plasmid pKD13 as a template, using commercial PCR reagents, a DNA fragment was obtained by PCR amplification, and purified for later use.

[0032] 2) Cultivate Escherichia coli BL21(DE3) in LB medium at 37 overnight, prepare competent cells by calcium chloride method, transform pKD46, coat ampicillin-resistant L...

Embodiment 2

[0048] Example 2 Glucose is used as carbon source to ferment and produce N-acetylneuraminic acid

[0049] 1. Seed and fermentation medium (1L):

[0050] (NH4) 2 SO 4 4g / L; KH 2 PO 4 6g;K 2 HPO 4 ·3H 2 O 8g; Yeast extract (purchased from OXOID company) 5g; Peptone 10g / L; Glucose 5g.

[0051] Feed solution: 500g / L glucose.

[0052] 2. Fermentation process:

[0053] 1) Pick a single CASOV-2 colony and place it in a 4ml LB test tube, and culture it at 37°C for 8-12 hours.

[0054] 2) Inoculate 2ml of primary seeds into 200ml of seed culture medium and culture at 37°C for 8-10 hours.

[0055] 3) Secondary seeds are inoculated in a fermenter with a liquid volume of 3.5 L at 37° C., stirring speed is 300-800 rpm, dissolved oxygen is kept above 30%, and pH is controlled at 6.9 with ammonia water.

[0056] 4) After the glucose is exhausted, add glucose at a rate of 5g / L.h.

[0057] 5) OD of fermentation broth 600 =25~30 o'clock, add IPTG (IPTG final concentration is 0.2mM)...

Embodiment 3

[0059] Example 3 Glycerol is used as carbon source to ferment and produce N-acetylneuraminic acid

[0060] Replace glucose with glycerol of 20g / L, other components of fermentation medium are the same as embodiment 2, and feeding medium is 600g / L glycerol;

[0061] The fermentation process is the same as in Example 2.

[0062] Finally, after about 80 hours of fermentation, the concentration of the product N-acetylneuraminic acid can reach more than 70 g / L.

[0063] The above culture results show that the genetically engineered bacteria constructed by the metabolic engineering technology have the ability of high-yield N-acetylglucosamine, and have the potential of industrialization.

[0064]

[0065]

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Abstract

The invention discloses a high-yield N-acetylneuraminic acid metabolic engineering bacterium and a construction method and application. According to the engineering bacterium, the activity of coded UDP-N-acetylglucosamine epimerase and N-acetylneuraminic acid synthetase gene enzyme is enhanced by expressing the two enzymes in Escherichia coli through a T7 strong promoter, and the recombinant Escherichia coli is constructed by removing genes of an N-acetylneuraminic acid decomposition pathway enzyme in the Escherichia coli; and a constructed engineered strain utilizes glucose or glycerinum as a substrate for fermentation cultivation to generate N-acetylneuraminic acid. The engineering bacterium utilizes glucose or glycerinum for synthesizing the N-acetylneuraminic acid, so that the fermentation level is high, the accumulated by-products are few, and the engineering bacterium has the industrial potential.

Description

technical field [0001] The invention relates to a high-yielding N-acetylneuraminic acid metabolic engineering bacterium and its construction method and application, belonging to the technical field of bioengineering. Background technique [0002] N-acetylneuraminic acid (Neu5Ac) is the main component of sialic acid, its content accounts for more than 99% of the entire sialic acid family, and it is usually located at the end of non-reducing oligosaccharides such as glycoproteins and glycolipids in the form of α-glycosides . N-acetylneuraminic acid exists widely in various organisms and plays a variety of physiological roles. In the human body, it can mainly improve the intestinal absorption of vitamins and minerals, anti-virus and improve the development of the nervous system of infants and young children. [0003] N-acetylneuraminic acid is used in the fields of food, health products and pharmaceutical intermediates due to its physiological functions and special structure....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P19/26C12R1/19
Inventor 柳鹏福孙立洁袁丽霞陈祥松王纪吴金勇朱薇薇王刚史吉平姚建铭
Owner 武汉中科光谷绿色生物技术有限公司
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