Recombinant escherichia coli and construction method and application thereof

A technology for recombining Escherichia coli and Escherichia coli, which is applied in the field of bioengineering, can solve the problems of limited production level, difficulty in obtaining and accumulating substrate L-fucose, etc., to eliminate adverse effects, facilitate intracellular synthesis and accumulation, The effect of weakening the glycolytic process

Pending Publication Date: 2021-03-30
HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

From the perspective of the synthesis route, the remedial synthesis route is relatively simple, but the required substrate L-fucose is extremely difficult to obtain, and the market price is relatively high, so this scheme is not yet suitable for large-scale GDP-L-fucose sugar preparation
[0006] In wild...

Method used

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  • Recombinant escherichia coli and construction method and application thereof
  • Recombinant escherichia coli and construction method and application thereof
  • Recombinant escherichia coli and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Construction of recombinant Escherichia coli using sorbitol to high-yield GDP-L-fucose

[0039] The method of using metabolic engineering and genetic engineering to construct recombinant Escherichia coli that utilizes sorbitol to produce high GDP-L-fucose, such as figure 1 shown, including the following steps:

[0040] The first step: Knock out the undecenylglucose phosphotransferase gene wcaJ in the original Escherichia coli JM109 (DE3), and construct Escherichia coli JM109 (DE3)△wcaJ to prevent the intracellular synthesis of Escherichia coli GDP-L- Fucose continues to transform downstream to generate dissanclein, which promotes the synthesis and accumulation of GDP-L-fucose.

[0041] Among them, the gene knockout method adopts the method of combining the CRISPR / Cas9 editing system and the traditional λ-Red homologous recombination system. During the operation, there is no need to introduce an exogenous resistance selection marker gene to replace the origina...

Embodiment 2

[0066] Example 2 Effect Test of Recombinant Escherichia coli Fermentatively Synthesizing GDP-L-Fucose

[0067] (1) Fermentative synthesis of GDP-L-fucose using recombinant Escherichia coli

[0068] Using the recombinant Escherichia coli constructed in Example 1 to ferment and synthesize GDP-L-fucose, the preparation work is as follows:

[0069] 1. The fermentation strains are: (1) Escherichia coli JM109(DE3)pR; (2) Engineering strain JM109(DE3)△W / pR; (3) Engineering strain JM109(DE3)△W / pRpE-D; (4) Engineering strain JM109(DE3)△W / pRpE-DAB; (5) Engineering strain JM109(DE3)△W / pRpE-DABgn; (6) Engineering strain JM109(DE3)△WP / pRpE-DABgn; As the test strain, the initial strain of Escherichia coli JM109 (DE3) was used as the control strain.

[0070] 2. Seed medium LB (g / L): tryptone 10, yeast extract 5, sodium chloride 10, pH 7.2-7.4; when preparing solid medium, add 17g / L agar powder.

[0071] 3. Initial fermentation medium (g / L): glucose 10-20, peptone 5-10, yeast powder 3-10, ...

Embodiment 3

[0087] Example 3 Construction and application of engineering strains producing 2'-fucosyllactose

[0088] A method for constructing an engineering strain producing 2'-fucosyllactose by means of metabolic engineering and genetic engineering, comprising the steps of:

[0089] According to the wbgL sequence of the α-1,2-fucosyltransferase gene in Escherichia coli (GenBank: KY499613.1), the α-1,2-fucosyltransferase gene was obtained through codon optimization and chemical synthesis The optimized sequence of wbgL, the optimized amino acid sequence is shown in SEQ ID NO.31. Using wbgL-F / R as a primer pair (SEQ ID NO.29 and SEQ ID NO.30), the wbgL gene fragment was amplified by PCR, and inserted into the vector pCDFDuet-1 through the restriction site NdeI / AvrII to obtain the recombinant plasmid pCDF -wbgL, whose structure is as Figure 4 shown. The recombinant plasmid pCDF-wbgL was co-transformed with the recombinant plasmids pRSF-manC-manB-gmd-wcaG and pET-srlD-pntAB-gsK-ndK into...

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Abstract

The invention belongs to the technical field of bioengineering, and relates to construction of genetic engineering strains, in particular to recombinant escherichia coli and a construction method andapplication thereof. According to the recombinant escherichia coli, an undecylenyl phosphate glucose phosphotransferase gene wcaJ is knocked out, manB, manC, gmd, wcaG and srlD genes are recombined and expressed, a pntAB gene, an ndK gene and a gsK gene are further recombined and expressed, and a pfkA gene is knocked out, so that a synthesis path of forming fructose 6-phosphate is enhanced and theglycolysis process of 6-fructose phosphate is weakened, the accumulation of fructose 6-phosphate is facilitated, and thus more precursor substances are provided for the synthesis of GDP-L-fucose; inaddition, the regeneration of the cofactors NADPH and GTP is enhanced, so that the capacity of producing GDP-L-fucose by the recombinant escherichia coli strain is further improved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to the construction of genetically engineered bacterial strains, in particular to a recombinant Escherichia coli and its construction method and application. Background technique [0002] Guanosine 5'-diphosphate-L-fucose (GDP-L-fucose), as an L-fucose donor, is essential in the biosynthesis of fucosylated oligosaccharides substrate. However, the low production level and high cost of GDP-L-fucose limit its application in the large-scale production of fucosyl oligosaccharides. [0003] In the prior art scheme, two pathways have been identified for the biosynthesis of the donor substrate GDP-L-fucose: the de novo pathway and the salvage pathway. [0004] The de novo synthesis pathway (De novo pathway) mainly originates from the central metabolic activity of bacteria. The synthesis pathway of GDP-L-fucose mainly starts from the glycolysis intermediate fructose 6-phosphate, and p...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/54C12N15/61C12N15/60C12N15/53C12P19/32C12P19/00C12R1/19
CPCC12N15/52C12N9/1288C12N9/90C12N9/1241C12N9/88C12N9/0006C12N9/1229C12N9/1205C12N9/0036C12N9/1051C12N15/70C12P19/32C12P19/00C12Y207/08031C12Y504/02008C12Y207/07013C12Y402/01047C12Y101/01271C12Y101/0114C12Y207/04006C12Y207/01011C12Y106/01001C12Y207/01073C12Y204/01069
Inventor 倪志坚陈祥松吴金勇廖迎雪李忠奎袁丽霞李翔宇王纪姚建铭
Owner HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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