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Construction method and application of high-yield lactoyl-N-tetrasaccharide microorganism

A lactoyl and galactosyl technology, applied in the field of microorganism construction, can solve the problems of low yield of lactoyl-N-tetrasaccharide, inability to meet the requirements of industrialized large-scale production and the like

Pending Publication Date: 2021-11-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the yield of lacto-N-tetrasaccharide synthesized by microbial methods currently used is low, and the reported β-1,3-galactosyltransferase may be a limiting factor, and none of them can meet the requirements of large-scale industrial production. , therefore, in order to solve the current bottleneck of microbial production, it is necessary to create more efficient production strains

Method used

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  • Construction method and application of high-yield lactoyl-N-tetrasaccharide microorganism
  • Construction method and application of high-yield lactoyl-N-tetrasaccharide microorganism
  • Construction method and application of high-yield lactoyl-N-tetrasaccharide microorganism

Examples

Experimental program
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preparation example Construction

[0030] 4. Preparation of competent E. coli: TAKARA kit.

[0031] 5. Culture medium and detection method for lacto-N-tetrasaccharide fermentation:

[0032](1) LB liquid medium: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L.

[0033] (2) LB solid medium: 10g / L peptone, 5g / L yeast extract powder, 10g / L sodium chloride, 15g / L agar powder.

[0034] (3) 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.

[0035] (4) HPLC detection conditions: high-efficiency ion-exchange chromatography; chromatographic column: CarboPac PA10 (4mm×250mm); detector: pulsed amperometr...

Embodiment 1

[0036] Embodiment 1: Construction of recombinant vector

[0037] The specific steps of recombinant expression vector construction are as follows (see Table 1 for the primer sequences involved):

[0038] (1) glmM, glmU-glmS, and lgtA gene (the nucleotide sequences of glmM, glmU, glmS, and lgtA are shown in SEQID NO.1-4 respectively) fragment acquisition and plasmid pRSF-(29)glmM-(29 The construction of )glmU-glmS and pET-(T7)lgtA is described in the patent whose publication number is CN111979168A.

[0039] (2) Obtaining of wbgO and galE gene fragments and construction of plasmid pCD-wbgO-galE:

[0040] wbgO was synthesized by Suzhou Jinweizhi after codon optimization. The nucleotide sequence of the wbgO gene fragment is shown in SEQ ID NO.7. Using the synthesized gene as a template and using WbgO-F / R as primers, wbgO was amplified by PCR Gene fragment, gel recovery DNA fragment; With the genome of Escherichia coli K-12 (Escherichia coli) as template, with WbgO-GalE-F / R as pri...

Embodiment 2

[0048] Embodiment 2: Construction of recombinant bacterial strain

[0049] Knockout the gene wecB encoding UDP-N-acetylglucosamine-2-epimerase WecB (NCBI sequence number YP_026253.1) and NagB encoding glucosamine-6 ​​phosphate deaminase (NCBI sequence number YP_026253.1) in Escherichia coli BL21 It is the gene nagB of NP_415204.1) and the gene lacZ of encoding β-galactosidase LacZ (NCBI sequence number is NP_414878.1), and the recombinant plasmid pRSF-(29 )glmM-(29)glmU-glmS and pET-(T7)lgtA, gene knockout and recombinant plasmid transfer method refer to the patent with publication number CN111979168A, to construct a recombinant large intestine producing lactyl-N-triose II Bacillus E10-WNL.

[0050] (1) The recombinant plasmid pCD-wbgO-galE constructed in Example 1 was transformed into Escherichia coli E10-WNL to construct a recombinant strain ELO1.

[0051] (2) The recombinant plasmid pCD-pf-galE constructed in Example 1 was transformed into Escherichia coli E10-WNL to cons...

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Abstract

The invention discloses a construction method and application of a high-yield lactoyl-N-tetrasaccharide microorganism, and belongs to the field of microbial genetic engineering. According to the method, a strain which is constructed in an early stage and is used for efficiently producing a precursor substance lactoyl-N-trisaccharide II is used as an original strain, and a key gene for synthesizing lactoyl-N-tetrasaccharide is over-expressed, so that the strain has the synthesis capability of producing lactoyl-N-tetrasaccharide. An efficient beta-1, 3-galactosyl transferase gene is screened, the coexpression beta-1, 3-galactosyl transferase gene and a UDP-glucose 4 epimerase gene (galE) which is a key gene for strengthening a UDP-galactose pathway are reasonably designed on a carrier pCDFDuet-1, so that the synthesis of the lactoyl-N-tetrasaccharide is improved, and in a shake flask experiment, the yield of the lactoyl-N-tetrasaccharide is increased, and the yield of the lactoyl-N-tetrasaccharide is increased. In a bottle shaking experiment, the capacity of producing the lactoyl-N-tetrasaccharide by the escherichia coli is 3.04 g / L, the yield of the lactoyl-N-tetrasaccharide in a 3L fermentation tank reaches 25.49 g / L, and the construction method has an industrial application prospect.

Description

technical field [0001] The invention relates to a construction method and application of a microorganism with high lactoyl-N-tetrasaccharide production, and belongs to the field of microbial genetic engineering. Background technique [0002] Human milk oligosaccharides (HMOs) have been shown to have unique physiological functions such as regulating the balance of intestinal microbiota in infants, promoting early brain development of newborns, and improving immunity. Lactoyl-N-tetrasaccharide, one of the main components of HMOs, is one of the twenty important core structures of HMOs. With it as the core unit, various breast milk oligosaccharides can be prepared through fucosylation and sialylation. Therefore, the efficient preparation of LNTs plays an important role in the large-scale synthesis of various HMOs. However, the current production costs of lactoyl-N-tetraose and lactoyl-N-neotetraose are relatively high and the production methods have certain limitations. In par...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/10C12N9/12C12N15/61C12N15/55C12N15/56C12N15/70C12P19/00C12R1/19
CPCC12N9/1051C12N9/2471C12N9/1241C12N9/1029C12N9/1096C12N9/78C12N9/90C12N15/70C12P19/26C12P19/18C12Y207/07023C12Y203/01003C12Y206/01016C12Y305/99006C12Y501/03014C12Y302/01023C12P19/04C12Y204/01102C12N2800/101
Inventor 沐万孟张文立朱莺莺李泽宇
Owner JIANGNAN UNIV
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