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An Escherichia coli engineering strain that efficiently produces gdp-fucose

A technology of Escherichia coli and fucose, which is applied in the fields of microbial metabolic engineering and genetic engineering, can solve the problems of metabolic burden and flux imbalance, and can not achieve yield, so as to relieve metabolic pressure, have important industrial application value, and increase accumulation. Effect

Active Publication Date: 2021-07-27
JIANGNAN UNIV
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  • Description
  • Claims
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Problems solved by technology

[0009] Furthermore, in the above examples, researchers did not perform modular regulation of the de novo synthesis pathway, but only considered a single variable and regulated the entire metabolic pathway in a linear fashion (simple overexpression of enzymes involved in the GDP-fucose synthesis pathway), Either single enhances NADPH regeneration or GTP regeneration, which may lead to metabolic burden and flux imbalance, unable to achieve global optimal yield

Method used

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  • An Escherichia coli engineering strain that efficiently produces gdp-fucose
  • An Escherichia coli engineering strain that efficiently produces gdp-fucose
  • An Escherichia coli engineering strain that efficiently produces gdp-fucose

Examples

Experimental program
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Effect test

Embodiment 1

[0050] Embodiment 1: Construction of recombinant vector

[0051] The specific steps are as follows (the construction process can refer to figure 2 ):

[0052] The genes manB, manC, gmd, wcaG, gsk and zwf encoding ManB, ManC, Gmd, WcaG, Gsk and Zwf in Escherichia coli MG1655 were passed through primers NdeI-manB-F / R, NcoI-manC-F / R, NcoI- gmd-F / R, NdeI-wcaG-F / R, NcoI-gsk-F / R and NdeI-zwf-F / R (see Table 1 for primer sequences) were amplified by PCR and recovered from DNA fragment gel to obtain relevant target gene fragments (PCR system see Table 2, PCR electrophoresis results see image 3 ). manB, gmd, and gsk were cloned into the first multiple cloning site of the corresponding Duet-1 plasmid after single-enzyme digestion (see Table 3 for the single-enzyme digestion system, and Table 4 for the enzyme-linked system), while manC, wcaG, and zwf were cloned by single-enzyme Excise cloned into the second cloning site of the corresponding Duet-1 plasmid, and finally obtained plas...

Embodiment 2

[0067] Example 2: Replacement of the original ribosome binding site on the expression plasmid

[0068] In addition to expressing the ribosome binding site (RBS-ori) of the plasmid itself, the present invention has selected two RBSs, one is the standard ribosome binding site (RBS-32) reported in the literature, and the other is manB, manC, gmd and wcaG have their own wild-type ribosome binding site (RBS-WT) on the genome, and replace the corresponding RBS into the original expression vector, thereby regulating the protein translation intensity of each target gene (for different RBS sequences, see table 5).

[0069] Table 5 RBS sequence

[0070]

[0071] Using the constructed plasmids pACYC-manC-manB and pET-gmd-wcaG as templates, primers manC-[RBS-32]-F / R, manC-[RBS-WT]-F / R, manB-[RBS- 32]-F / R, manB-[RBS-WT]-F / R, gmd-[RBS-32]-F / R, gmd-[RBS-WT]-F / R, wcaG-[RBS-32] -F / R, wcaG-[RBS-WT]-F / R obtained the corresponding fragments and vectors (see Table 6 for primer sequences), an...

Embodiment 3

[0081] Embodiment 3: the knockout of Escherichia coli BL21 (DE3) genome gene

[0082] The specific steps are as follows (the specific operation process can refer to Figure 4 ):

[0083] (1) Use primers ΔwcaJ-upflank-F / R and ΔwcaJ-downflank-F / R to amplify the upstream and downstream fragments of the wcaJ gene by PCR respectively (see Table 8 for PCR primer sequences);

[0084] (2) Use primers ΔwcaJ-upflank-F and ΔwcaJ-downflank-R to carry out fusion PCR amplification of the upstream and downstream fragments to obtain a complete template gene (for the results of nucleic acid gel electrophoresis of PCR amplification products, see Figure 5 );

[0085] (3) Use primer N20-F / R to perform PCR amplification on the existing pTargetF plasmid to obtain the pTargetF plasmid with targeting wcaJ;

[0086] (4) transfer the pCas plasmid into Escherichia coli BL21 (DE3) by means of electroporation, and add 10 mM arabinose to induce the expression of the λ-Red Escherichia coli gene recombin...

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Abstract

The invention provides an Escherichia coli production strain for efficiently producing GDP-fucose, and belongs to the technical fields of genetic engineering and microbial metabolism engineering. The present invention uses glucose as a substrate to regulate phosphomannose mutase (ManB), GDP-mannose pyrophosphorylase (ManC) and GDP-mannose 4,6-dehydratase (Gmd) in the metabolic pathway through combination and GDP-fucose synthase (WcaG) expression to relieve the metabolic stress of the GDP-fucose synthesis pathway. The present invention also knocks out the gene UDP-glucose-lipid carrier transferase (WcaJ) that decomposes GDP-fucose in Escherichia coli through the CRISPR / Cas9 gene editing system and strengthens the regeneration of cofactors in the metabolic pathway to further increase intracellular GDP-fucose Accumulation of fucose.

Description

technical field [0001] The invention relates to an Escherichia coli engineering strain for efficiently producing GDP-fucose, and belongs to the technical fields of genetic engineering and microbial metabolism engineering. Background technique [0002] GDP-fucose is a nucleotide sugar that is widely used as a sugar donor and a metabolic intermediate in various organisms, and is an essential substance in the regulation of life metabolism. GDP-fucose is mainly used as an important glycosyl donor for protein glycosylation, and is a precursor for the synthesis of other sugar nucleotides in eukaryotes, and is mainly involved in the synthesis of fucosylated oligosaccharides in prokaryotes Biosynthesis. [0003] Human milk oligosaccharides (HMOs) are among the most abundant solid components in human milk. Its biological functions, such as improving intestinal flora, inhibiting pathogen adhesion, regulating immune response and promoting brain development, hold great promise for inf...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/90C12N15/61C12N15/60C12N15/54C12N15/53C12P19/32C12R1/19
CPCC12N9/0006C12N9/1205C12N9/1288C12N9/88C12N9/90C12N15/70C12N15/902C12P19/32C12Y101/01049C12Y101/01271C12Y207/08031C12Y402/01047C12Y504/02008
Inventor 沐万孟张文立万李朱莺莺李雯
Owner JIANGNAN UNIV
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