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Recombinant corynebacterium glutamicum for accumulating N-acetylneuraminic acid and application thereof

A technology of acetylneuraminic acid and Corynebacterium glutamicum, applied in the field of genetic engineering, achieves good application prospects, easy to use, and simple construction methods

Active Publication Date: 2018-07-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the whole cell transformation method needs to add N-acetylglucosamine as a synthetic precursor, and most methods still need to add pyruvate as a synthetic precursor

Method used

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  • Recombinant corynebacterium glutamicum for accumulating N-acetylneuraminic acid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Construction of recombinant plasmids

[0029] According to the acetylglucosamine isomerase coding gene (age) sequence in Anabaena sp.CH1 published on NCBI, the age gene was synthesized by GenScript Biotechnology Co., Ltd., and the primer age-F:5' was designed -TTCACACAGGAAACAGAATTCGAAGGAGTCTTCACATGGGCAAAAACTTACAAGCTCT-3', age-R:5'-CATTCTACTCTGACTTATGAAAGTGCTTCAAACTGTTGC-3'. The above primers were used to amplify the acetylglucosamine isomerase encoding gene (age) using the synthesized age gene fragment as a template.

[0030]According to the N-acetylneuraminic acid synthase coding gene (neuB) sequence in Escherichia coli K-1 published on NCBI, the neuB gene was synthesized by GenScript Biotechnology Co., Ltd., and the primer neuB-F was designed : 5'-TCATAAGTCAGAGTAGAATGAGAAGGAGTAGATTCATGTCTAACATTCTACATCGTGGC-3', neuB-R: 5'-CATCCGCCAAAACAGAAGCTTGTTAACTTTATTCTCCTGGTTTTAAATTCGC-3'. The above primers were used to amplify the gene encoding N-acetylneuraminic acid...

Embodiment 2

[0037] Example 2 Knockout of the gene cg2937 encoding the N-acetylneuraminic acid transporter

[0038] According to the upstream and downstream sequences of the N-acetylneuraminic acid transporter gene (cg2937) in Corynebacterium glutamicum ATCC 13032 published on NCBI, the primer cg2937-U-F: 5'-CTATGACATGATTACGAATTCGCTGTGAGCTTTGATGGTTTC-3'; cg2937- U-R: 5′-ACATTGATCTCTACTCTGACTGCCGGTGTTGTCTGGTGCA-3′; cg2937-D-F: 5′-GTCAGAGTAGAGATCAATGTCGAATCCTACGACCAGGTACA-3′; cg2937-D-R: 5′-TGCCTGCAGGTCGACTCTAGAGGTGATTGGGGTGATCAGC-3′. Using the genome of Corynebacterium glutamicum ATCC13869 (purchased from the American Type Microorganism Collection) as a PCR template, the upstream homolog of the N-acetylneuraminic acid transporter gene (cg2937) was amplified using primers cg2937-U-F and cg2937-U-R. Source arm sequence △cg2937-Up; use primers cg2937-D-F and cg2937-D-R to amplify the downstream homology arm sequence △cg2937-Down of the N-acetylneuraminic acid transporter gene (cg2937). △cg293...

Embodiment 3

[0039] Example 3 Blocking of N-acetylneuraminic acid intracellular catabolic pathway

[0040] By knocking out the genes encoding N-acetylmannokinase nanK, the genes encoding N-acetylmannose-6-phosphate isomerase nanE, the genes encoding acetylglucosamine-6-phosphate deacetylase nagA and glucosamine on the chromosome -6-phosphate deaminase encoding gene nagB, to block the catabolism of N-acetylneuraminic acid in the cell.

[0041] According to the upstream and downstream sequences of the N-acetylneuraminic acid operon (5'-nagB-nagA-nanA-nanK-nanE-3') in Corynebacterium glutamicum ATCC 13032 published on NCBI, primers NEU- U-F: 5'-CTATGACATGATTACGAATTCGATTTCGGGGAGACATTCACT-3'; NEU-U-R: 5'-GTACCTGAGAATGTAGTTTTTTGGTGCCAACGCGATCATC-3'; NEU-D-F: 5'-AAAACTACATTCTCAGGTACAAACGCTGATCACTACCGTCT-3'; NEU-D-R: 5'-TGCCC-GCAGGTA'. Using the genome of Corynebacterium glutamicum ATCC13869, purchased from the American Type Microorganism Collection as a PCR template, the N-acetylneuraminic acid ...

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Abstract

The invention discloses recombinant corynebacterium glutamicum for accumulating N-acetylneuraminic acid and application thereof and belongs to the field of genetic engineering. The recombinant corynebacterium glutamicum takes corynebacterium glutamicum as an expression host, and an N-acetylneuraminic acid synthesis way is reinforced through over-expression of a glucosamine-fructose-6 phosphate aminotransferase gene, a glucosamine acetylase encoding gene, a phosphatase encoding gene, an acetylglucosamine isomerase encoding agene and an N-acetylneuraminic acid synthase encoding gene; an N-acetylneuraminic acid transport protein encoding gene in the corynebacterium glutamicum and a related gene in an intracellular N-acetylneuraminic acid decomposition, utilization and metabolism are knocked off to obtain corynebacterium glutamicum genetically engineered bacteria for extracellularly accumulating the N-acetylneuraminic acid and the yield reaches 110mg / L; a foundation is laid for further modifying corynebacterium glutamicum through metabolic engineering to produce the N-acetylneuraminic acid.

Description

technical field [0001] The invention relates to a recombinant corynebacterium glutamicum accumulating N-acetylneuraminic acid and an application thereof, belonging to the field of genetic engineering. Background technique [0002] As the most important compound molecule in sialic acid, N-acetylneuraminic acid is an important food nutritional additive and a new drug precursor. Its effects include promoting brain development of infants, maintaining healthy brain function of the elderly and enhancing the body's immunity. At present, the industrial production of N-acetylneuraminic acid is mainly produced by chemical extraction and whole-cell transformation methods. Due to the low total content of N-acetylneuraminic acid in natural raw materials (egg yolk, bird's nest and bovine colostrum, etc.), the extraction and separation process is complicated, resulting in low yield and high cost of traditional extraction methods. Therefore, the production of N-acetylneuraminic acid by wh...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P19/26C12R1/15
CPCC12N9/1085C12N9/1096C12N9/16C12N9/90C12P19/26C12Y205/01056C12Y206/01016C12Y305/01108C12Y504/02003
Inventor 陈坚堵国成王淼刘延峰董迅衍
Owner JIANGNAN UNIV
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