Recombinant escherichia coli for synthesizing 2 '-fucosyllactose by using mannose and application of recombinant escherichia coli

A technology of fucosyllactose and Escherichia coli, applied in the field of microbial metabolic engineering, can solve the problems of low utilization rate of carbon source, high cost, no industrial production, etc., and achieve the effect of improving utilization rate of carbon source and conversion rate.

Pending Publication Date: 2022-04-05
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The remedial approach needs to use fucose as a carbon source to produce 2′-fucosyllactose. Due to the high cost of using fucose as a carbon source, it basically does not have the potential for industrial production, and the 2′-fucosyllactose The de novo synthesis pathway can use glycerol or glucose as a carbon source to synthesize GDP-fucose and then synthesize 2′-fucosyllactose, which has the possibility of industrial production, but due to the weak synthesis pathway of 2′-fucosyllactose, it is difficult Competes with the glycolytic pathway, resulting in a large amount of carbon source being consumed by cell growth or metabolism, and the carbon source utilization rate for the production of 2′-fucosyllactose is low, resulting in a low level of 2′-fucosyllactose production

Method used

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  • Recombinant escherichia coli for synthesizing 2 '-fucosyllactose by using mannose and application of recombinant escherichia coli
  • Recombinant escherichia coli for synthesizing 2 '-fucosyllactose by using mannose and application of recombinant escherichia coli
  • Recombinant escherichia coli for synthesizing 2 '-fucosyllactose by using mannose and application of recombinant escherichia coli

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] The construction of embodiment 1 chassis bacterium bacterial strain L

[0089] Strain E.coli K12 MG1655ΔlacIZ::P trc - Build of wcaG-gmd-lacy

[0090] Taking Escherichia coli MG1655 as the original strain, the P in the lac operon sequence was knocked out lac Promoter sequence and lacI, lacZ gene, and use P on the lacI, lacZ gene locus trc The promoter overexpresses wcaG, gmd, and lacy genes. P trc The promoter is P trp promoter and P lac The spliced ​​promoter of the promoter has a ratio of P lac Promoter higher transcription efficiency.

[0091] The method used to construct this strain is λRed recombination. The main purpose is to construct a two-step homologous recombination fragment, and use pKD46 (GenBank: MF287367) as a homologous recombination plasmid to carry out gene knockout and integration. The homologous recombination fragment in the first step includes upstream and downstream homology arms, chloramphenicol resistance gene and sacB gene. The second-...

Embodiment 2

[0104] Embodiment 2: the construction of bacterial strain L1 (knockout manA gene)

[0105] The strain L1 was constructed, and the strain L constructed in Example 1 was used as the chassis strain, and the manA (phosphomannose isomerase) gene on the genome was knocked out.

[0106] The method used to construct this bacterial strain is λRed recombination technology. The main purpose is to construct a two-step homologous recombination fragment, and use pKD46 (GenBank: MF287367) as a homologous recombination plasmid to carry out gene knockout and integration. The homologous recombination fragment in the first step includes upper and lower homology arms, chloramphenicol resistance gene and sacB gene. The second-step homologous recombination fragment contains upstream and downstream homology arms.

[0107] The specific method is described in detail below:

[0108] 1. The first step is the construction of the homologous recombination fragment up-cat-sacB-down. Construct the cat-sa...

Embodiment 3

[0118] Embodiment 3: Construction of bacterial strain L2 (knockout wcaj gene)

[0119] Construct strain L2, use strain L1 as the chassis strain, knock out the UDP-glucolipid carrier transferase coding gene (Wcaj) on the genome, and use cas9 to construct this strain. It is mainly to construct the homologous recombination fragment in the first step, including the upstream and downstream homology arms, chloramphenicol resistance gene and N20 gene (SEQ ID NO.15), and then electroporate into the chassis strain that needs to be knocked out. After the selection medium with different resistances grew out, the Escherichia coli that was successfully recombined in the first step was induced to express the Pcago plasmid to produce cas9 protein to screen the fragments that were successfully recombined. The specific method is described in detail below:

[0120]1. The first step is the construction of the homologous recombination fragment up-cat-N20-down. The cat-N20 gene fragment was cons...

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Abstract

The invention relates to a method for constructing recombinant escherichia coli, synthesizing GDP-fucose by using mannose, further producing 2 '-fucosyllactose and improving the utilization rate of mannose, and belongs to the field of microbial metabolism engineering. The engineering bacterium takes escherichia coli as a starting strain, a Plac promoter sequence and lacI and lacZ genes in a lac operon sequence are knocked out, wcaG, gmd and lacy genes are overexpressed on 1acI and lacZ gene loci, then a phosphomannose isomerase coding gene manA is knocked out on a genome, a phosphomannose mutase coding gene manB and alpha-(1, 2, 3, 4, 5, 6, 7, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8, 8 and (2) obtaining a fucosyl transferase coding gene futC and a mannose-1-phosphate guanine transferase coding gene manC. According to the fermentation strategy for producing the 2 '-fucosyllactose through the de novo synthesis route of the 2'-fucosyllactose, the utilization rate of a carbon source for producing the 2 '-fucosyllactose through escherichia coli fermentation can be greatly increased.

Description

Technical field: [0001] The invention relates to a method for constructing 2'-fucosyllactose-producing recombinant Escherichia coli, which has improved mannose utilization efficiency and belongs to the field of microbial metabolism engineering. Background technique: [0002] Human milk oligosaccharides (HMOs) are a unique class of carbohydrates that naturally occur in breast milk and have many benefits for human health. At present, people focus on their application as food additives. [0003] 2′-fucosyllactose (2′-FL) is the largest human milk oligosaccharide (HMO) product (accounting for about 31%), which can be utilized by beneficial microbial flora in the intestinal tract to regulate intestinal and adding 2'-FL to infant milk powder can enhance the immunity of newborn babies and effectively enhance the physique of newborn babies; 2'-FL also plays a role in brain development, neuron transmission and synapse formation, so Adding 2'-FL to the diet can promote brain developm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/53C12N15/54C12N15/56C12N15/60C12N15/61C12N15/31C12P19/24C12P19/18C12P19/00C12R1/19
Inventor 李庆刚李玉李浩正郑艳玲宗剑飞肖卫华彭则涛程亚楠李畅路福平
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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