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Genetically engineered bacteria for producing N-acetylneuraminic acid as well as construction method and application thereof

A technology of acetylneuraminic acid and genetically engineered bacteria, which is applied to the genetically engineered bacteria producing N-acetylneuraminic acid and the fields of its construction and application, can solve the problem of low productivity of Neu5Ac, influence of catalytic reaction efficiency and conversion rate, and difficulty in scale. It is easy to industrialize large-scale production, reduce substrate transport barriers, and improve catalytic efficiency.

Active Publication Date: 2014-07-16
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method does not need to separate and purify the enzyme. At the same time, due to the protection of the cell membrane, the enzyme is relatively more stable and the cofactor is easy to regenerate.
However, since two kinds of bacteria expressing different enzymes need to be cultivated separately, and the bacteria are concentrated and then mixed, this mode is difficult to industrialize. At the same time, the substrate and product enter and exit the cell many times and are blocked by the cell wall, thus affecting the catalytic reaction efficiency and transformation. Rate
[0004] It can be seen from the above several methods for synthesizing N-acetylneuraminic acid that the existing production methods have low output, high cost, and difficulty in scale expansion, resulting in low productivity of Neu5Ac, which is difficult to meet future market demand.

Method used

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  • Genetically engineered bacteria for producing N-acetylneuraminic acid as well as construction method and application thereof
  • Genetically engineered bacteria for producing N-acetylneuraminic acid as well as construction method and application thereof
  • Genetically engineered bacteria for producing N-acetylneuraminic acid as well as construction method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0066] Embodiment 1, construct the genetically engineered bacterium that produces N-acetylneuraminic acid

[0067] 1. Construction of recombinant plasmids for co-expression of N-acetylglucosamine 2-isomerase and N-acetylneuraminic acid aldolase

[0068] 1. Construction of recombinant plasmid pEcNA

[0069] Using the genome of luminescent cyanobacteria Anabaena sp. The coding gene of isomerase, that is, the age gene), the fragment size is about 1200bp, which is consistent with the target fragment. After sequencing analysis, the results show that the amplified sequence is the same as the age gene sequence numbered BA000019.2 on NCBI, and the age gene The nucleotide sequence is shown in sequence 7 in the sequence listing, and the amino acid sequence of N-acetylglucosamine 2-isomerase encoded by the nucleotide sequence is shown in sequence 1 in the sequence listing.

[0070] After the age gene was digested with EcoRI and HindⅢ, the age gene fragment was recovered; the pBADhisB v...

Embodiment 2

[0118] Embodiment 2, Utilize the preparation of N-acetylneuraminic acid-producing genetically engineered bacteria to produce N-acetylneuraminic acid

[0119] 1. Induction of genetically engineered bacteria producing N-acetylneuraminic acid

[0120]Self-induction culture: the genetically engineered bacteria TL004, TL005, TL006, TL007, TL008, TL002, TL003 and pEcNA / K12 of the metabolic engineering bacteria producing N-acetylneuraminic acid were streaked onto agar containing 1.5% concentration by mass percentage and containing 50μg / mL ampicillin LB plate, cultured at 37°C for 12h. Pick the single clone grown on the plate, inoculate it into the liquid LB medium containing 80 μg / mL ampicillin, culture overnight at 37°C with shaking at 250 rpm; In self-inducing medium ZYM, shake culture at 37° C., rotation speed 250 rpm, and culture time 16 h.

[0121] The formula of self-induction medium ZYM is as follows: 100mL A+2mL B+2mL C+200μL D+100μL E (the following are mass percentage con...

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Abstract

The invention discloses genetically engineered bacteria for producing N-acetylneuraminic acid as well as a construction method and application thereof. A method for preparing the genetically engineered bacteria for producing N-acetylneuraminic acid comprises the following step of: introducing genes associated with the N-acetylneuraminic acid synthesis into host bacteria so as to obtain recombinant bacteria for producing N-acetylneuraminic acid, wherein the genes associated with the N-acetylneuraminic acid synthesis are coding genes of N-acetylglucosamine and 2-isomerase and a coding gene of N-nacetylneuraminic acid aldolase. The genetically engineered bacteria for producing N-acetylneuraminic acid constructed by the invention have the following advantages that the used raw materials are relatively cheap and liable to realize industrial mass product, so that the genetically engineered bacteria play an important role in the production of N-acetylneuraminic acid in the future.

Description

technical field [0001] The invention relates to a genetically engineered bacterium producing N-acetylneuraminic acid and its construction method and application. Background technique [0002] N-acetylneuraminic acid (N-acetyl-D-neuraminic acid, Neu5Ac) is the most representative member of the sialic acid family, a pyranose composed of 9 carbon atoms, has a wide range of biological functions, in the treatment of Influenza, neurological diseases, inflammation and tumors have important medical value, and have broad application prospects and high market value, especially in the prevention and treatment of influenza (including avian influenza H5N1 and 2009 H1N1). good effect. [0003] At present, the sources of N-acetylneuraminic acid are limited, and there are few studies on industrial production. The traditional production methods lead to its high price, which greatly limits the related research and development and application. N-acetylneuraminic acid can be extracted from na...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12R1/19
Inventor 林白雪张子娟陶勇
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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