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A kind of genetically engineered bacteria and production method for improving lactoyl-n-trisaccharide II yield

A technology encoding gene, acetylglucosamine, applied in microorganism-based methods, biochemical equipment and methods, glycosylases, etc., can solve the problem of low yield of lactoyl-N-trisaccharide II and inability to achieve industrialized large-scale production and other problems to achieve the effect of relieving metabolic stress

Active Publication Date: 2022-07-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the yield of lactoyl-N-triose II synthesized by the microbial method currently used is low, which cannot meet the requirements of large-scale industrial production. Therefore, in order to solve the current bottleneck of microbial production, it is necessary to create a more efficient production strain of

Method used

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  • A kind of genetically engineered bacteria and production method for improving lactoyl-n-trisaccharide II yield
  • A kind of genetically engineered bacteria and production method for improving lactoyl-n-trisaccharide II yield
  • A kind of genetically engineered bacteria and production method for improving lactoyl-n-trisaccharide II yield

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0041] Preparation of E. coli competent: TAKARA kit.

[0042] Lactoyl-N-trisaccharide II fermentation process and detection:

[0043] LB liquid medium: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L.

[0044] LB solid medium: 10g / L peptone, 5g / L yeast extract powder, 10g / L sodium chloride, 15g / L agar powder.

[0045] Fermentation medium: 20g / L glucose, 13.5g / L potassium dihydrogen phosphate, 4.0g / L diammonium hydrogen phosphate, 1.7g / L citric acid, 1.4g / L magnesium sulfate heptahydrate and 10ml / L trace metal elements; Trace metal elements include: 10g / L ferrous sulfate, 2.25g / L zinc sulfate heptahydrate, 1.0g / L anhydrous copper sulfate, 0.35g / L manganese sulfate monohydrate, 0.23g / L sodium borate decahydrate, 0.11g / L ammonium molybdate, 2.0g / L calcium chloride dihydrate.

[0046] (1) Fermentation process of lactoyl-N-trisaccharide II: the constructed strain was inoculated into LB liquid medium at 37°C, 200 rpm, and cultured overnight for 12 h to obtain seed liqui...

Embodiment 1

[0048] Example 1: Construction of recombinant vectors

[0049] The specific steps for the construction of the recombinant expression vector are as follows (see Table 1 for the primer sequences involved):

[0050] (1) Obtainment of glmM, glmU-glmS gene fragments: using the genome of Escherichia coli K-12 (Escherichia coli) as a template, using glmM-F / glmM-R as primers, PCR amplifies the glmM gene fragment, and the DNA is recovered by gel The fragments were ligated into the first multiple cloning site (MCS1) of the vectors pRSFDuet-1, pETDuet-1 and pCDFDuet-1 by a seamless cloning kit (Nanjing Novozymes Life Technology Co., Ltd.); then glmUS-F / The glmUS-R was used as the primer, and the glmU-glmS gene cluster fragment was amplified by PCR. The DNA fragment was recovered by gel, and the gene was ligated into the second polyclonal vector pRSFDuet-1, pETDuet-1 and pCDFDuet-1 by single digestion with kpnI. Site (MSC2), the final plasmids are pRSF-glmM-glmU-glmS, pCDF-glmM-glmU-glm...

Embodiment 2

[0054] Example 2: Replacing the original ribosome binding site on the expression plasmid

[0055] In the present invention, in addition to the ribosome binding site (RBS T7) of the expression plasmid itself, two RBSs (RBS 29 and RBS 31) of different intensities reported in the literature were selected (see Table 2 for different RBS sequences). RBS 29 (representing strong regulation), RBS T7 (representing moderate regulation), and RBS 31 (representing weak regulation) were selected to regulate the protein translation strength of each target gene.

[0056] Table 2 RBS sequences

[0057]

[0058] Using the constructed plasmids pRSF-glmM-glmU-glmS and pET-lgtA as templates, use primers 29lgtA-F / R, 31lgtA-F / R, 29M-F / R, 31M-F / R, 29US-F / R , 31US-F / R, to obtain the corresponding fragments (see Table 3 for primer sequences), and assemble the fragments using the One Step Cloning Kit (Vazyme) to obtain the corresponding recombinant plasmids (see Table 4 for plasmid information). It ...

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Abstract

The invention discloses a genetically engineered bacterium and a production method for improving the yield of lactoyl-N-trisaccharide II, belonging to the field of microbial genetic engineering. The present invention controls the expression of glmM, glmU, glmS and lgtA in the synthetic pathway of lactyl-N-trisaccharide II in combination, thereby accurately regulating the carbon flux of the metabolic pathway, relieving the metabolic pressure, and knocking out the lactyl-N-Escherichia coli host The expression of wecB, nagB and lacZ in the synthesis pathway of trisaccharide II further increased the yield of lactyl-N-trisaccharide II. L was increased to 4.82g / L, and in the 3L fermenter, the yield of lactoyl-N-trisaccharide II reached 46.2g / L, which has the prospect of industrial application.

Description

technical field [0001] The invention relates to a genetically engineered bacterium and a production method for improving the yield of lactoyl-N-trisaccharide II, belonging to the field of microbial genetic engineering. Background technique [0002] Breast milk is generally considered to be the most important source of nutrition for infants. As the third most abundant solid component in breast milk, the synthesis of breast milk oligosaccharides plays an important role in the growth of beneficial intestinal flora in infants and prevention of the adhesion of pathogenic bacteria to epithelial cells. There are more than 200 types of breast milk oligosaccharides that have been reported, mainly divided into three categories, sialylated, fucosylated and afucosylated neutral breast milk oligosaccharides, accounting for 12%- 14%, 35%-50% and 42-55%. Among them, the afucosylated neutral breast milk oligosaccharides with the highest proportion mainly include lactoyl-N-tetrasaccharide ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N9/90C12N9/12C12N9/10C12N9/38C12N9/78C12P19/00C12R1/19
CPCC12N9/90C12N9/1096C12N9/1241C12N9/1051C12N9/78C12N9/2471C12N15/70C12P19/00C12Y501/03014C12Y206/01016C12Y207/07023C12Y204/01146C12Y504/0201C12Y305/99006C12Y302/01023
Inventor 沐万孟张文立朱莺莺万李
Owner JIANGNAN UNIV
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