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Recombinant bacillus subtilis capable of increasing yield of N-acetylneuraminic acid

A technology of Bacillus subtilis and acetylneuraminic acid, which is applied in the field of genetic engineering, can solve the problems of insufficient metabolic flux of Bacillus subtilis, affecting the metabolism and synthesis of N-acetylneuraminic acid, and achieves a simple construction method and good application prospects , Ease of use

Active Publication Date: 2017-07-07
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the anabolic flux of Bacillus subtilis is insufficient, affecting the metabolic synthesis of N-acetylneuraminic acid

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 host cell construction

[0029] 1) Construction of recombinant fragments

[0030] By fusion PCR, the amino acid sequence of the glucosamine acetylase coding gene GNA1 clone fragment of SEQ ID NO.2 is fused with the recombinant homology arm and the zeocin resistance gene;

[0031] 2) Construction of recombinant plasmids

[0032] The cloned amino acid sequence is the coding gene AGE of N-acetylglucosamine isomerase of SEQ ID NO.3, and the coding gene NeuB of N-acetylneuraminic acid synthase of SEQ ID NO.4 is connected to the recombinant expression on plasmid pP43NMK;

[0033] 3) Construction of recombinant Bacillus subtilis producing N-acetylneuraminic acid

[0034] Transform the recombinant fragment in the above step 1) into Bacillus subtilis (Bacillus subtilis 168ΔnagP ΔnagP ΔgamP ΔgamA ΔnagAΔnagBΔ1dh Δpta::lox72), and recombine it into the genome to obtain a recombinant Bacillus subtilis engineering bacterium, named B6C; then the above step 2) The recombi...

Embodiment 2

[0035] The construction of embodiment 2 recombinant plasmids

[0036]According to the phosphotransferase system glucose-specific enzyme EIICBA component coding gene ptsG published on NCBI, its upstream 914bp homology arm sequence was designed, and the designed sequences were primers of SEQ ID NO.5 and SEQ ID NO.6: ptsG-1F: 5'-TAAAAGACGAGAAGGAACAAAAGCAG-3', ptsG-1R: 5'-TCCTGTGTGAAATTGTTATCCGCTCAAGAATTGACCTCCTCTTTTTACTAGTCTG-3'; design its downstream 927bp homology arm sequence, and design primers whose sequences are SEQ ID NO.7 and SEQ ID NO.8 respectively: ptsG-2F : 5'-CGTCGTGACTGGGAAAACCCCTGGCGGGGTGTTAGTACGCCGTGCTTGT-3', ptsG-2R: 5'-AGATGTGCGTCCGCCGATAT-3'; in Bacillus subtilis.AppliedandEnvironmental Microbiology 74,5556-5562, doi:10.1128 / aem.01156-08(2008)) sequence information to amplify the Spectinomycin resistance gene, the designed sequences are SEQ ID NO.9 and SEQ ID NO.10 respectively Primers: Spc-F: 5'-TAGTAAAAAAGAGGAGGTCAATTCTTGAGCGGATAACAATTTCACACAGG-3', Spc-R: 5'...

Embodiment 3

[0037] The construction of embodiment 3 recombinant Bacillus subtilis

[0038] The constructed recombinant knockout fragment was transformed into Bacillus subtilis (Bacillus subtilis 168ΔnagPΔnagPΔgamPΔgamAΔnagAΔnagBΔ1dhΔpta::lox72; Δctc::p43-Gna1, pP43NMK-AGE-NeuB). Spc-F and Spc-R primers were used to select transformants for colony PCR, and a 1264bp band appeared, verifying that the recombinant Bacillus subtilis was successfully constructed.

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Abstract

The invention discloses recombinant bacillus subtilis capable of increasing the yield of N-acetylneuraminic acid, and belongs to the field of genetic engineering. Supply of phosphoenolpyruvic acid in the synthesis pathway of N-acetylneuraminic acid is increased by taking bacillus subtilis (bacillus subtilis 168 delta nagP delta nagP delta gamP delta gamA delta nagA delta nagB delta 1dh delta pta::lox72; delta ctc::p43-Gna1, pP43NMK-AGE-NeuB) as an expression host and knocking out ptsG of a glucose specific enzyme EIICBA component of a phosphotransferase system, and therefore the synthesis passway is enhanced; compared with an original strain, the recombinant bacillus subtilis has the advantages that the yield of N-acetylneuraminic acid is increased to 660 mg / L from 190 mg / L, and a foundation is laid for improving N-acetylneuraminic acid production from bacillus subtilis in metabolic engineering.

Description

technical field [0001] The invention relates to a recombinant bacillus subtilis for increasing the output of N-acetylneuraminic acid, which belongs to the field of genetic engineering. Background technique [0002] N-acetylneuraminic acid is a kind of sugar substance in organisms, which widely exists in microorganisms and mammals. In the human body, N-acetylneuraminic acid is a key substance for cell information transmission and participates in multiple physiological processes such as cell recognition, signal transduction, and fertilization. Therefore, N-acetylneuraminic acid is widely used in anti-inflammation, enhancing infant immunity, promoting infant brain development, and maintaining brain health in the elderly. At present, N-acetylneuraminic acid is mainly extracted from relatively rich natural materials such as casein and bird’s nest. The products obtained are likely to cause allergic reactions, or they are synthesized by chemical methods under harsh conditions, and...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12P19/26A23L33/135C12R1/125
CPCC12N9/1205C12N15/75C12P19/26C12Y207/01
Inventor 陈坚堵国成刘延峰张晓龙
Owner JIANGNAN UNIV
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