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Genetically engineered bacterium for producing fucosyllactose and production method

A technology of fucosyllactose and genetically engineered bacteria, applied in the fields of metabolic engineering and food fermentation, can solve the problem of low yield of fucosyllactose, and achieve the effects of cheap substrate, fast growth of strains, and efficient synthesis

Pending Publication Date: 2022-05-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to solve the problem of low yield of fucosyllactose synthesized by existing biological methods, the present invention provides a genetically engineered bacterium producing 2'-FL and / or 3-FL and its construction method

Method used

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  • Genetically engineered bacterium for producing fucosyllactose and production method
  • Genetically engineered bacterium for producing fucosyllactose and production method
  • Genetically engineered bacterium for producing fucosyllactose and production method

Examples

Experimental program
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preparation example Construction

[0056] Preparation of Escherichia coli Competent: Kit of Shanghai Sangon Bioengineering Company.

[0057] LB liquid medium: 10g / L peptone, 5g / L yeast extract, 10g / L sodium chloride.

[0058] LB solid medium: 10g / L peptone, 5g / L yeast extract, 10g / L sodium chloride, 18g / L agar powder.

[0059] Fermentation medium: glycerol 30g / L, potassium dihydrogen phosphate 13.5g / L, citric acid 1.7g / L, diammonium hydrogen phosphate 4.0g / L, magnesium sulfate heptahydrate 1.4g / L, yeast extract 10g / L, Trace metal solution 10mL / L (ferric citrate 10g / L, magnesium sulfate heptahydrate 2.25g / L, copper sulfate pentahydrate 1.0g / L, manganese sulfate monohydrate 0.35g / L, borax 0.23g / L, molybdic acid Ammonium 0.11g / L, calcium chloride dihydrate 2.0g / L), pH 6.8.

[0060] Determination of 2'-FL and 3-FL:

[0061] HPLC measurement: 1 mL of fermentation broth was boiled at 100°C for 10 min, centrifuged at 12000 r / min for 5 min, the supernatant was filtered through a 0.22 μm membrane, and the production ...

Embodiment 1

[0063] Example 1: Transformation of Escherichia coli chassis microorganisms by CRISPR-Cas9 gene knockout technology

[0064] According to the metabolic pathway of fucosyllactose ( figure 1 shown), using Escherichia coli BL21(DE3)ΔlacZΔwcaJ as the starting strain (the construction method of Escherichia coli BL21(DE3)ΔlacZΔwcaJ has been disclosed in the patent literature with publication number CN112342176A), using the CRISPR-Cas9 gene knockout system separately or simultaneously The nudD, nudK, pfkA, pfkB, mtlD, lon, fsaA, fsaB, and fucIK genes in the genome of Escherichia coli BL21(DE3)ΔlacZΔwcaJ were knocked out, and the specific steps were as follows (see Table 1 for the primer sequences involved):

[0065] (1) Take the nudD gene as an example, find the specific target gRNA (20bp) of the target gene through http: / / www.regenome.net / cas-offinder, use nudD-gRNA-F / gRNA-R upstream and downstream primers, PCR amplification was performed using pTargetF plasmid (Addgene: #62226) as...

Embodiment 2

[0076] Example 2: Construction of de novo synthesis pathway and batch fermentation of knockout strains to produce fucosyllactose

[0077] The specific steps for the construction of the expression vector for the de novo synthesis pathway are as follows (see Table 2 for the primer sequences involved):

[0078] (1) Obtaining manB, manC, gmd and wcaG fragments: using the genome of Escherichia coli K12 as a template, using primers manCB_F / R and GW_F / R for PCR amplification, gel recovery DNA, and obtaining manC-manB, gmd-wcaG genes fragment.

[0079] (2) Using pRSFDuet-1, pETDuet-1, pCDFDuet-1, pACYCDuet-1 and pCOLADuet-1 as templates, use primers V1_F / R and V2_F / R to amplify the vector backbone sequence. According to the In-Fusion cloning technique, the fragments manC-manB were respectively inserted between the Nco I / BamH I restriction sites of the above-mentioned vector, and the fragment gmd-wcaG were respectively inserted between the NdeI / Xho I restriction sites of the above-men...

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Abstract

The invention relates to a genetically engineered bacterium for producing fucosyllactose and a production method of the genetically engineered bacterium, and belongs to metabolic engineering and food fermentation technologies. According to the invention, a synthetic biological means is utilized, an escherichia coli cell factory of fucosyllactose is constructed based on a de novo synthetic route, bypass related genes lacZ, wcaJ, nudD, pfkA and lon in host cells are knocked out by adopting a CRISPR / Cas9 gene editing technology, and genes manB, manC, gmd and wcaG are assembled through a multi-plasmid expression system, so that the fucosyllactose is obtained. Constructing expression vectors with different copy numbers to increase the expression level of key genes; fucosyltransferase from different sources is screened, so that the activity of rate-limiting enzyme is improved. The constructed recombinant escherichia coli can be used for synthesizing 2 '-fucosyllactose and 3-fucosyllactose by utilizing glycerol, is stable in heredity and high in expression level, and has obvious industrial production potential.

Description

technical field [0001] The invention relates to a genetic engineering bacterium producing fucosyllactose and a production method, belonging to metabolic engineering and food fermentation technology. Background technique [0002] Human milk oligosaccharides (Human Milk Oligosaccharides, HMOs) is the third largest solid component in human milk after lactose and fat. As an important immune active component in breast milk, HMOs play a vital role in the health, growth and development of infants, and more and more animal and clinical trials have confirmed the beneficial properties of HMOs, such as maintaining intestinal ecology as a prebiotic Balance, anti-pathogen adhesion, immune regulation, and promotion of nervous system development and repair and other functional activities. Fucosyllactose, including 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL), is the most abundant neutral human milk oligosaccharide secreted in human milk, accounting for about 35% of the total, ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/53C12N15/54C12N15/56C12N15/57C12N15/60C12N15/61C12P19/00C12R1/19
CPCC12N9/2471C12N9/1288C12N9/1205C12N9/2402C12N9/0006C12N9/52C12N9/88C12N9/90C12N9/1051C12N15/70C12N15/52C12P19/00C12Y207/08031C12Y302/01023C12Y207/01011C12Y101/01017C12Y401/02017C12Y503/01025C12Y207/01052C12Y504/02008C12Y101/01132C12Y101/01271C12Y204/01065C12Y204/01069
Inventor 张涛李梦丽江波李晨晨胡苗苗
Owner JIANGNAN UNIV
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