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Escherichia coli engineering strain for efficiently producing 2'-fucosyllactose

A technology of fucosyllactose and Escherichia coli, applied in the fields of synthetic biology and microbial metabolic engineering, can solve the problems of short salvage pathway, low 2′-FL yield, and influence on host metabolism, etc.

Active Publication Date: 2021-07-30
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the problem that the heterologous expression of the target enzyme has low enzyme activity and affects the host metabolism, resulting in low 2'-FL production, the present invention knocks out Escherichia coli Endogenous genes related to the catabolism of the substrates L-fucose and lactose and the key intermediate product GDP-L-fucose, to improve the metabolic pathway of L-fucose → 2′-FL, by strengthening the relationship with 2 '-FL synthesis-related genes, and improve the binding efficiency and stability of intracellular enzyme complexes through short peptides, the rescue pathway is shorter and the bifunctional enzyme Fkp can be soluble expressed in E. coli, so it can be more effective Increased production of 2′-FL by efficiently supplying GDP-L-fucose precursor

Method used

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  • Escherichia coli engineering strain for efficiently producing 2'-fucosyllactose
  • Escherichia coli engineering strain for efficiently producing 2'-fucosyllactose
  • Escherichia coli engineering strain for efficiently producing 2'-fucosyllactose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1: Construction of recombinant Escherichia coli knockout lacZ

[0079] (1) Use primer N20-ΔlacZ-F / R to perform PCR amplification on the existing pTargetF plasmid to obtain the pTargetF plasmid with the targeting lacZ gene;

[0080] (2) Use primers ΔlacZ-UH-F / R and ΔlacZ-DH-F / R to amplify upstream and downstream homologous fragments of the lacZ gene by PCR (see Table 6 for PCR primer sequences);

[0081] (2) These two homologous fragments pass The II one-step cloning kit was connected to the linearized pTargetF-ΔlacZN20 vector (the vector was linearized by primer pTargetT-F / R amplification) to construct the plasmid pTargetT-ΔlacZN20;

[0082] (3) Transfer the pCas plasmid into E.coliBL21(DE3) by electroporation, and add 20mM arabinose to induce the expression of the λ-Red Escherichia coli gene recombination system;

[0083] (4) Electroporate the above-mentioned Escherichia coli BL21(DE3) with the pTargetT-ΔlacZN20 plasmid, spread it on an LB plate containing ka...

Embodiment 2

[0086] Example 2: Construction of Escherichia coli expressing futC and fkp heterologously

[0087] The genes futC (nucleotide sequence shown in SEQ ID NO.2) and fkp (nucleotide sequence shown in SEQ ID NO.1) were synthesized by Shanghai Sangon Bioengineering Co., Ltd. The genes futC and fkp were respectively amplified by primers futC-F / R(NcoI) and fkp-F / R(NdeI) and then inserted into the NcoI restriction site of the first multiple cloning site of pETDuet-1 after restriction enzyme ligation point and the NdeI restriction site of the second multiple cloning site, constituting the plasmid pET-futC-fkp (see Table 1 for the primer sequence, and refer to the plasmid construction flow chart figure 2 (1)).

[0088] The plasmid pET-futC-fkp was transformed into E.coliBL21(DE3)ΔlacZ, and the strain containing plasmid pET-futC-fkp was obtained after sequencing verification, which was named WLS01.

Embodiment 3

[0089] Embodiment 3: the construction of the recombinant escherichia coli of knocking out fucIK, araA, rhaA, wcaJ gene

[0090] According to the method of Example 1, the fucIK gene in the bacterial strain WLS01 was knocked out, and the bacterial strain WLS02 was constructed;

[0091] According to the method of Example 1, the araA gene in the bacterial strain WLS02 was knocked out, and the bacterial strain WLS03 was constructed;

[0092] According to the method of Example 1, the rhaA gene in the bacterial strain WLS02 was knocked out, and the bacterial strain WLS04 was constructed;

[0093] According to the method of Example 1, the rhaA gene in the bacterial strain WLS03 was knocked out, and the bacterial strain WLS05 was constructed;

[0094] According to the method in Example 1, the wcaJ gene in the strain WLS05 was knocked out to construct the strain WLS06.

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Abstract

The invention provides an escherichia coli engineering strain for efficiently producing 2'-fucosyllactose, and belongs to the technical field of synthetic biology and microbial metabolism engineering. According to the invention, glycerol is taken as a carbon source, lactose and L-fucose are taken as substrates, and self-assembled oligopeptide-mediated pathway key enzymes Fkp and FutC are introduced to assemble and combine into an enzyme compound, so that the loss of metabolic intermediate products is reduced, the production efficiency of the products is improved, and the production yield and yield of the products 2'-FL are improved. In addition, a series of genes related to substrate degradation and intermediate product shunting in escherichia coli are knocked out through a CRISPR / Cas9 gene editing system, and meanwhile cyclic regeneration of cofactors in a metabolic pathway is enhanced, so that the yield of 2'-FL is further increased. The obtained escherichia coli engineering strain can accumulate 30.5 g / L of 2'-FL after being fed for 64 hours in batches under the condition of a 3L fermentation tank, and the molar yield is 0.661 mol(2'-FL) / mol fucose and 0.495 mol(2'-FL) / mol lactose. The invention has important significance on industrial production of the 2'-FL.

Description

technical field [0001] The invention relates to an Escherichia coli engineering strain for efficiently producing 2′-fucosyllactose, belonging to the technical fields of synthetic biology and microbial metabolism engineering. Background technique [0002] Human milk oligosaccharides (HMOs) are the third most abundant solid component in breast milk, which play a very important role in infant growth and development. Studies have shown that HMO has prebiotic effects, which can promote the proliferation of intestinal probiotics such as bifidobacteria and inhibit the growth of harmful bacteria, thereby regulating the intestinal flora. In addition, HMOs are able to bind pathogenic bacteria and affect the barrier function of the gut, thereby modulating local and systemic immunity. In HMOs, the proportion of fucosylated oligosaccharides is about 50%. Among them, 2′-FL is the most abundant and most common fucosylated HMOs. 2'-FL can act as a potent inhibitor of bacterial or viral a...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P19/00C12R1/19
CPCC07K14/195C07K14/245C12N9/1051C12N9/1205C12N9/1229C12N9/1235C12N15/70C12P19/00C12Y207/04008C12Y207/06004Y02A50/30
Inventor 沐万孟张文立万李朱莺莺
Owner JIANGNAN UNIV
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