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Recombinant bacteria for high-yield production of N-acetylneuraminic acid by utilizing manual double carbon sources

An acetylneuraminic acid, artificial technology, applied in the field of genetic engineering, can solve the problems of not being able to use glucose and sodium gluconate at the same time, limiting the industrial production of N-acetylneuraminic acid, etc., so as to improve the synthesis efficiency and improve the N-acetylneuraminic acid. Amino acid synthesis, easy to use effect

Active Publication Date: 2018-08-24
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Sodium gluconate is a cheap carbon source that can be efficiently utilized by Bacillus subtilis, and can supplement the supply of N-acetylneuraminic acid synthesis precursor phosphoenolpyruvate through the pentose phosphate pathway, but when the substrate In the presence of glucose, under the mediation of carbon source catabolite repression, Bacillus subtilis cannot simultaneously utilize glucose and sodium gluconate (see the paper "Bacillus subtilis GntR regulation modified to devise artificialtransient induction systems" published in 2016), therefore, Limiting the industrial production of N-acetylneuraminic acid

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 host cell construction

[0036] 1) Construction of recombinant integration fragments

[0037] By fusion PCR, the amino acid sequence is the phosphoenolpyruvate carboxykinase coding gene yqaB clone fragment of SEQ ID NO: 2, the recombinant homology arm, and Spectinomycin resistance gene and P 43 Promoter gene fusion;

[0038] By fusion PCR, the chloromycetin resistance and base sequence are the promoter P of SEQ ID NO:4 43 Fragment, glutamine-fructose-6-phosphate transaminase gene glms recombination homology arm fusion;

[0039] 2) Construction of recombinant plasmids

[0040] The cloned amino acid sequence is the coding gene AGE of N-acetylglucosamine isomerase of SEQ ID NO:5, and the coding gene NeuB of N-acetylneuraminic acid synthase of SEQ ID NO:6 is connected to the recombinant expression On plasmid pP43NMK;

[0041] 3) Construction of recombinant Bacillus subtilis producing N-acetylneuraminic acid

[0042] The phosphoenolpyruvate carboxykinase codi...

Embodiment 2

[0043] The construction of embodiment 2 recombinant plasmids

[0044] The upstream homology arm primers were designed according to the gluconate kinase gene gntK promoter sequence published on NCBI: gntK-1F: 5'-CGTGTTGATAGACGGCATTACTGTCTCGGGAAAT-3', gntK-1R: 5'-CCTGTGTGAAATTGTTATCCGCTCACACTCACCTTCCTCACTCAAGGAGTATACTTGT-3';

[0045] Design the downstream homology arm primer: gntK-2F:

[0046] 5'-TAGGTAAGAGAGGAATGTACACATGACTAGTTATATGTTAGGAATCGATATCGGCACGA-3', gntK-2R: 5'-GGGCCATCCATGCATTCAGTTAACG-3';

[0047] Will P 43 The promoter was inserted into the P7S6 plasmid (the construction method of the P7S6 plasmid was disclosed in Yan, X., Yu, H.J., Hong, Q. & Li, S.P.Cre / lox system and PCR-based genome engineering in Bacillus subtilis. Applied and Environmental Microbiology 74, 5556-5562, doi:10.1128 / aem.01156-08 (2008)), constituting the p7SP43 plasmid. According to the P7SP43 plasmid sequence, design spectinomycin resistance and P 43 Promoter amplification primer: SpcP 43 -F...

Embodiment 3

[0048] The construction of embodiment 3 recombinant Bacillus subtilis

[0049] The constructed recombinant integration fragment was transformed into Bacillus subtilis (Bacillus subtilis 168ΔnagPΔnagPΔgamPΔgamAΔnagAΔnagBΔ1dhΔptaΔptsG::lox72; Δctc::p43-Gna1; ΔspsC::p43-yqaB; Δglms::p43-glms, pP43NMK-AGE-NeuB). Using SpcP 43 -F and SpcP 43 -R primers were used to select transformants for colony PCR, and a 1499bp band appeared, verifying that the recombinant Bacillus subtilis was successfully constructed and named B6CG3.

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Abstract

The invention discloses recombinant bacteria for high-yield production of N-acetylneuraminic acid by utilizing manual double carbon sources and belongs to the field of genetic engineering. A gluconicacid kinase gene gntK and gluconic acid permease gntP are overexpressed through a strong promoter P43, and the manual double carbon sources including glucose and sodium gluconate are used for fermenting to realize efficient supply of phosphoenolpyruvic acid in an N-acetylneuraminic acid synthesis way and the synthesis way is strengthened. Compared with a starting strain, recombinant bacillus subtilis has the advantage that the yield of N-acetylneuraminic acid is improved to 1.52g / L from 1.02g / L; a foundation is laid for producing N-acetylneuraminic acid by carrying out further metabolic engineering modification on the recombinant bacillus subtilis.

Description

technical field [0001] The invention relates to a recombinant bacterium for high-yielding N-acetylneuraminic acid by using artificial double carbon sources, which belongs to the field of genetic engineering. Background technique [0002] N-acetylneuraminic acid is an important carbohydrate substance in organisms, which is responsible for signal transmission and other functions in organisms. In the human body, N-acetylneuraminic acid participates in important physiological processes such as intercellular signal transduction and recognition. Therefore, N-acetylneuraminic acid is widely used in anti-inflammation of influenza, enhancing immunity, promoting brain development of school-age infants, and maintaining brain function and health of the elderly. At present, N-acetylneuraminic acid is mainly extracted from relatively abundant natural materials such as eggs and bird’s nest, and the obtained products are likely to cause allergic reactions, or are synthesized by chemical me...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12P17/06
CPCC12N9/00C12N9/1205C12N15/75C12P17/06C12Y207/01012
Inventor 陈坚堵国成刘延峰王淼张晓龙
Owner JIANGNAN UNIV
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