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Genetically engineered bacteria capable of increasing yield of lactoyl-N-trisaccharide II and production method for genetically engineered bacteria

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: 2020-11-24
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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  • Genetically engineered bacteria capable of increasing yield of lactoyl-N-trisaccharide II and production method for genetically engineered bacteria
  • Genetically engineered bacteria capable of increasing yield of lactoyl-N-trisaccharide II and production method for genetically engineered bacteria
  • Genetically engineered bacteria capable of increasing yield of lactoyl-N-trisaccharide II and production method for genetically engineered bacteria

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

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

[0042] Lactoyl-N-triose 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) Lactoyl-N-triose II fermentation process: Inoculate the constructed strain in LB liquid medium, 37°C, 200rpm, and culture overnight for 12h to obtain seed liquid, and insert the seed liquid...

Embodiment 1

[0048] Embodiment 1: Construction of recombinant vector

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

[0050] (1) Acquisition 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 fragments, and recovers DNA from the gel The fragment was connected to the first multiple cloning site (MCS1) of the vectors pRSFDuet-1, pETDuet-1 and pCDFDuet-1 through the seamless cloning kit (Nanjing Novizan Life Science and Technology Co., Ltd.); glmUS-R is the primer, the glmU-glmS gene cluster fragment is amplified by PCR, the DNA fragment is recovered from the gel, and the gene is connected to the second polyclonal of the vector pRSFDuet-1, pETDuet-1 and pCDFDuet-1 by kpnI single enzyme digestion site (MSC2), the final plasmids obtained are pRSF-glmM-glmU-glmS, pCDF-glmM-glmU-glmS and ...

Embodiment 2

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

[0055] In addition to expressing the ribosome binding site (RBS T7) of the plasmid itself, the present invention also selects two RBS (RBS 29 and RBS 31) of different strengths reported in the literature (see Table 2 for different RBS sequences). In this embodiment Select RBS 29 (represents strong regulation), RBS T7 (represents moderate regulation), and RBS 31 (represents weak regulation) to regulate the protein translation intensity of each target gene.

[0056] Table 2 RBS sequence

[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, the corresponding fragments were obtained (see Table 3 for primer sequences), and the fragments were assembled using the One Step Cloning Kit (Vazyme) kit to obtain the corresponding recombinant plasmids (see Table 4 for plasmid i...

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Abstract

The invention discloses genetically engineered bacteria capable of increasing the yield of lactoyl-N-trisaccharide II and a production method, and belongs to the field of microbial genetic engineering. According to the invention, the expression of glmM, glmU, glmS and lgtA in a lactoyl-N-trisaccharide II synthetic pathway is regulated and controlled in a combined manner, so the carbon flux of a metabolic pathway is accurately regulated and controlled, and the metabolic pressure is relieved; the expression of wecB, nagB and lacZ in an escherichia coli host, namely the lactoyl-N-trisaccharide IIsynthetic pathway is knocked out, so the yield of the lactoyl-N-trisaccharide II is further increased; in a flask shaking experiment, the capacity of escherichia coli for producing the lactoyl-N-trisaccharide II is increased to 4.82 g / L from 0.53 g / L; in a fermentation tank with a volume of 3 L, the yield of the lactoyl-N-trisaccharide II reaches 46.2 g / L; and thus, thegenetically engineered bacteria have industrial application prospect.

Description

technical field [0001] The invention relates to a genetically engineered bacterium and a production method for increasing the yield of lactoyl-N-triose II, belonging to the field of microbial genetic engineering. Background technique [0002] Breast milk is generally considered the most important source of nutrition for infants. As the third 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 in preventing 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 non-fucosylated neutral breast milk oligosaccharides, and each accounted for 12%- 14%, 35%-50% and 42-55%. Among them, the non-fucosylated neutral human milk oligosaccharides mainly include lactoyl-N-tetraose (LNT) and lactoyl-N-neotetraose (LNnT). A large...

Claims

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Application Information

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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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