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Genetically engineered bacterium for producing 2'-fucosyllactose and application of genetically engineered bacterium

A technology of fucosyllactose and engineering bacteria is applied in the fields of metabolic engineering and food fermentation, which can solve the problems of expensive glycoside donors and low synthesis yield, and achieve the effect of wide industrial application value.

Pending Publication Date: 2021-02-09
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the existing problems of low synthesis yield of 2'-fucosyllactose and expensive glycoside donors, the present invention provides a method for transforming Escherichia coli by means of genetic engineering, knocking out the decomposition of 2'-fucosyllactose Metabolism-related genes strengthen the key synthetases of the de novo synthesis pathway, and the recombinant bacteria can efficiently produce 2'-fucosyllactose using cheap substrates, which has wide industrial application value

Method used

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  • Genetically engineered bacterium for producing 2'-fucosyllactose and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing 2'-fucosyllactose and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing 2'-fucosyllactose and application of genetically engineered bacterium

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Experimental program
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Effect test

Embodiment 1

[0049] Embodiment 1: Construction of the recombinant expression plasmid containing T7 promoter

[0050] The specific steps for constructing the recombinant expression vector are as follows (see Table 1 for the primer sequences involved):

[0051] (1) Obtaining manB, manC, gmd and wcaG fragments: using the genome of Escherichia coli K12 as a template, using primers manB_F1 / R1, manC_F1 / R1, gmd_F1 / R1 and wcaG_F1 / R1 to perform PCR amplification, gel recovery DNA, and obtain manB, manC, gmd and wcaG gene fragments.

[0052] (2) Using pRSFDuet-1, pETDuet-1, pCDFDuet-1, pACYCDuet-1 and pCOLADuet-1 empty plasmids as templates, use V1_F / R to amplify the vector backbone. According to the In-Fusion cloning technology, the manB fragment was connected with pRSFDuet-1, pETDuet-1, pCDFDuet-1, pACYCDuet-1 and pCOLADuet-1 respectively, and positive clones were screened and sequenced to obtain recombinant plasmids pRSFDuet-1-manB, pETDuet- 1-manB, pCDFDuet-1-manB, pACYCDuet-1-manB, and pCOLAD...

Embodiment 2

[0061] Embodiment 2: Construction of the recombinant expression plasmid containing Trc promoter

[0062] The specific steps of recombinant expression vector construction are as follows (see Table 2 for the primer sequences involved):

[0063](1) Construction of pRSFDuet-Trc, pETDuet-Trc, pCDFDuet-Trc, pACYCDuet-Trc and pCOLADuet-Trc vectors: using pTrcHis2B as a template, using primers Trc_F / R to amplify the Trc promoter and MCS region, using pRSFDuet-1, The pETDuet-1, pCDFDuet-1, pACYCDuet-1 and pCOLADuet-1 empty plasmids were used as templates, and the vector backbone sequence was amplified using primers VDuet_F / R. According to the In-Fusion cloning technique, the two fragments were ligated and transformed into DH5α, positive clones were screened, and an expression vector containing a Trc promoter was finally obtained.

[0064] (2) Obtaining manB, manC, gmd and wcaG fragments: using the genome of Escherichia coli K12 as a template, using primers manB_F2 / R2, manC_F2 / R2, gmd_...

Embodiment 3

[0074] Example 3: Knockout of the lacZ and wcaJ genes of Escherichia coli BL21 (DE3) based on the CRISPR-Cas9 gene editing system

[0075] The specific steps of gene knockout are as follows (see Table 3 for the primer sequences involved):

[0076] (1) Taking the lacZ gene as an example, design the gRNA of the lacZ gene through http: / / www.regenome.net / cas-offinder / , introduce the designed 20bp sequence into gRNAR, use gRNAF / gRNAR as the upstream and downstream primers, and the pTargetF plasmid is template for PCR amplification. The amplified product was digested with the restriction endonuclease Dpn I to remove the redundant circular pTargetF plasmid. The amplified product was transformed into Escherichia coli DH5α competent cells, the plasmid was extracted, and identified by primers gRNAPF / gRNAPR sequencing, and the successfully constructed knockout plasmid was named pTF-ΔlacZ.

[0077] (2) Using the Escherichia coli BL21 (DE3) genome as a template, the upstream homology arm...

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Abstract

The invention relates to a genetically engineered bacterium for producing 2'- fucosyllactose and application of the genetically engineered bacterium, and belongs to metabolic engineering and food fermentation technologies. The expression levels of phosphomannomutase, mannose-1-phosphoguanine transferase, GDP-mannose-6-dehydrogenase, GDP-fucosyl synthase, alpha-1,2-fucosyl transferase and lactose permease in a metabolic pathway are regulated and controlled by changing the expression of a transcription regulation factor in escherichia coli, so that an optimal plasmid combination is obtained; beta-galactosidase and UDP-glucose lipid carrier transferase in an escherichia coli synthetic route are knocked out through a CRISPR-Cas9 gene editing system, and therefore, the accumulated amount of 2'-fucosyllactose is increased; and the recombinant escherichia coli obtained by the invention can be used for synthesizing 2'-fucosyllactose by utilizing glycerol or glucose, is stable in heredity and high in expression level, and has obvious industrial production potential.

Description

technical field [0001] The invention relates to a genetically engineered bacterium producing 2'-fucosyllactose and an application thereof, belonging to metabolic engineering and food fermentation technology. Background technique [0002] Human milk oligosaccharides (HMOs) are the third largest solid component in breast milk after fat and lactose. Fucosylated HMOs, including 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose, lacto-N- New tetrasaccharides, lactyl-N-fucopentose, these substances act as natural anti-adhesion agents, by preventing the adhesion of pathogens, reducing the gastrointestinal symptoms caused by harmful bacteria such as Campylobacter jejuni, Escherichia coli and Streptococcus pneumoniae in infants and young children. The risk of infection of the tract, urogenital system and respiratory tract. 2'-FL is the most abundant human milk oligosaccharide in breast milk, accounting for about 30% of the total HMOs. Studies have shown that infa...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N9/90C12N9/38C12N9/12C12N9/10C12N9/04C12P19/00C12R1/19
CPCC12N15/52C12N9/2471C12N9/90C12N9/1288C12N9/0006C12N9/1241C12N9/1051C12N9/1205C12N15/70C12P19/00C12Y302/01023C12Y207/08031C12Y504/02008C12Y207/07013C12Y101/01132C12Y101/01271C12Y204/01069
Inventor 张涛江波李梦丽李晨晨
Owner JIANGNAN UNIV
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