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Genetically engineered bacteria for efficiently producing N-acetylglucosamine

A technology of genetically engineered bacteria and acetylamino, which is applied in the field of genetically engineered bacteria for efficient production of N-acetylglucosamine, can solve problems such as unstable enzyme activity of glucosamine 6-phosphate synthase, and achieve broad industrial application prospects and stability. Good, high fermentation yield

Inactive Publication Date: 2015-01-21
SHANGHAI RES & DEV CENT OF INDAL BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the N-acetylglucosamine transferase genes from different sources were screened, and the glucosamine-6-phosphate synthase mutant gene capable of high-yielding glucosamine was obtained by random screening by error-prone PCR, and was expressed on the chromosome of Escherichia coli K-12 strain On the basis of knocking out the nagDCABE gene cluster, the T7lac promoter-mediated glucosamine 6-phosphate synthase mutant gene glmS*54 and several copies of the N-acetylglucosamine transferase gene ScGNA1 from Saccharomyces cerevisiae were integrated. The results showed that the fermentation At 72 hours, the yield of N-acetylglucosamine of the genetically engineered bacteria containing two copies of ScGNA1 on the chromosome of Escherichia coli reached 110g / L; Defects on the decline in

Method used

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  • Genetically engineered bacteria for efficiently producing N-acetylglucosamine
  • Genetically engineered bacteria for efficiently producing N-acetylglucosamine
  • Genetically engineered bacteria for efficiently producing N-acetylglucosamine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 , Construction of recombinant plasmid pET28a-glmS

[0045] The wild-type glucosamine 6-phosphate synthase gene glmS is derived from Escherichia coli W3110, NCBI accession number: 89106884, and the size is 1830bp. Using the Escherichia coli W3110 genome as a template, use F-glmS-BsaI and R-glmS-BamHI primers to perform PCR amplification to obtain the glmS gene. After the amplified product is recovered, it is digested with BsaI and BamHI, and pET-28a(+) vector The plasmid was double-digested with NcoI and BamHI, and the above two fragments were ligated, transformed into E. coli DH5α competent cells and screened for kanamycin resistance to obtain the recombinant plasmid pET28a-glmS.

Embodiment 2

[0046] Example 2 , Construction of recombinant plasmid pET28a-mglmS

[0047] According to literature reports (Deng MD, Severson DK, Grund AD, et al. Metabolic engineering of Escherichia coli for industrial production of glucosamine and N-acetylglucosamine. Metabolic Engineering, 2005, 7:201-214), wild-type glucosamine 6-phosphate Synthetase activity is subject to feedback inhibition by the metabolite glucosamine-6-phosphate of this synthetic pathway, which is one of the key factors for the limited production of N-acetylglucosamine.

[0048] GlmS (A38T / R249C / G471S) is a mutant that can tolerate glucosamine inhibition and can increase the yield of the final product. Therefore, on the basis of cloning wild-type glmS, glmS was subjected to site-directed mutation to obtain glmS (A38T / R249C / G471S) mutant, named mglmS.

[0049] The experimental operation of site-directed mutagenesis at A38T site is as follows:

[0050]Using the recombinant plasmid pET28a-glmS as a template, the ...

Embodiment 3

[0052] Example 3 , Construction of recombinant plasmid pET28a-ScGNA1

[0053] N-acetylglucosamine transferase gene ScGNA1 is derived from Saccharomyces cerevisiae (baker's yeast), NCBI accession number: 4115732, with a size of 480bp. Using the Saccharomyces cerevisiae genome as a template, use F-ScGNA1-BsaI and R-ScGNA1-BamHI primers to perform PCR amplification to obtain the ScGNA1 gene. After the amplified product is recovered, it is double-digested with BsaI and BamHI and used for pET-28a(+) plasmid NcoI and BamHI double digestion, the above two fragments were ligated, transformed into Escherichia coli DH5α competent cells and screened for kanamycin resistance to obtain the recombinant plasmid pET28a-ScGNA1.

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Abstract

The invention provides genetically engineered bacteria for efficiently producing N-acetylglucosamine. The genetically engineered bacteria are prepared by the following steps: integrating chromosome deficiency nag DCABE gene clusters of Escherichia coli, respectively connecting 6-glucosamine phosphate synthetase mutant genes and N-acetylglucosamine transferase genes which are respectively mediated by a T7 promoter and a Trc promoter with a gene expression cassette in series, wherein the 6-glucosamine phosphate synthetase mutant genes are obtained by mutating wild 6-glucosamine phosphate synthetase genes from an Escherichia coli W3110 strain source into A38T / R249C / G471S mutants. The genetically engineered bacteria constructed by the invention have the advantages of high N-acetylglucosamine fermentation yield and high strain stability and have wide industrial application prospects.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a genetically engineered bacterium for efficiently producing N-acetylglucosamine. Background technique [0002] N-acetylglucosamine (N-Acetylglucosamine, GlcNAc), also known as 2-acetamide-2-deoxy-D-glucose (2-Acetamido-2-Deoxy-D-Dlucose), molecular formula: C 8 h 15 NO 6 , molecular weight: 221.0, melting point: 205°C, white crystalline powder, easily soluble in water, the structural formula is as follows: [0003] [0004] N-acetylglucosamine is the basic unit of many important polysaccharides in biological cells, especially the exoskeleton of crustaceans has the highest content. It is an important precursor for the synthesis of bifidus factors and has many important physiological functions in organisms. It is clinically used for the treatment of rheumatism and rheumatoid arthritis. It can also be used as a food antioxidant, a food additive for infants, an...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P19/26C12R1/19
CPCC12N15/52C12N9/1051C12N9/1096C12N9/14C12P19/26C12Y204/01155C12Y206/01016
Inventor 陶荣盛朱傅赟杨晟范文超柳鹏福陈成王祎沈正权丁鹏蒋宇
Owner SHANGHAI RES & DEV CENT OF INDAL BIOTECH
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