Saccharomyces cerevisiae engineering strain with high yield of fucosyllactose and application of saccharomyces cerevisiae engineering strain

A technology of fucosyllactose and Saccharomyces cerevisiae, which is applied in the field of genetic engineering, can solve the problems of difficulty in meeting industrial production, industrial production constraints, affecting the difficulty of product separation and purification, and product safety.

Active Publication Date: 2020-07-31
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Escherichia coli may produce endotoxin, which affects the difficulty of product separation and purification and product safety; in addition, Escherichia coli is easily contaminated by phages during fermentation, which restricts the industrial production of 2-FL by Escherichia coli
[0004] Saccharomyces cerevisiae is generally considered to be a safe microorganism. There have been studies using Saccharomyces cerevisiae to synthesize 2-FL; however, the yield of 2-FL synthesized by Saccharomyces cerevisiae strains in existing reports is only 0.5g / L, which is difficult to meet the needs of industrial production

Method used

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  • Saccharomyces cerevisiae engineering strain with high yield of fucosyllactose and application of saccharomyces cerevisiae engineering strain
  • Saccharomyces cerevisiae engineering strain with high yield of fucosyllactose and application of saccharomyces cerevisiae engineering strain
  • Saccharomyces cerevisiae engineering strain with high yield of fucosyllactose and application of saccharomyces cerevisiae engineering strain

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1: Construction of recombinant genetically engineered strain of Saccharomyces cerevisiae that can absorb lactose

[0028] The construction of the recombinant engineering strain in this example uses Saccharomyces cerevisiae W303-1a as the starting strain.

[0029] The total DNA of Kluyveromyces lactis was extracted with Omega kit, and the sequence of lactose permease was amplified from it using primers Lac12-F and Lac12-R, named Lac12, its amino acid The sequence is shown in SEQ ID No.1, and the nucleic acid sequence is shown in SEQ ID No.2.

[0030] Using the total DNA of Saccharomyces cerevisiae W303-1a as a template, using BamHI-GAL1-F and GAL1-LAC12-R as primers to amplify the gal1 promoter, using lac12-CYC1-F and CYC1-XhoI-R to amplify Generate cyc1 terminator, construct P by SOE-PCR gal1 -lac12-T cyc1 The expression cassette was seamlessly connected to the pRS304 vector after double digestion with BamHI and XhoI to obtain the recombinant vector pRS304-P gal1 -la...

Embodiment 2

[0036] Example 2: Construction of a recombinant Saccharomyces cerevisiae genetically engineered strain producing GDP-fucose

[0037] From Escherichia coli K12, the GDP-mannose dehydratase (GDP-mannose-4,6-dehydratase) sequence was amplified with primers GMD-F and GMD-R, named Gmd, and its amino acid sequence is as SEQ ID No. 3 is shown, and its nucleotide sequence is shown in SEQ ID No. 4. The sequence of GDP-L-fucose synthase (GDP-L-fucose synthase) was amplified with primers WcaG-F and WcaG-R, named WcaG, its amino acid sequence is shown in SEQ ID No. 5, and its nucleotides The sequence is shown in SEQ ID No.6.

[0038] The pUMRI-A plasmid (Ye, L., X.Lv, and H. Yu, Assembly of biosynthetic pathway in Saccharomyces cerevisiae using a marker recyclable integrative plasmidtoolbox.Frontiers of Chemical Engineering in China, 2017.11(1): p.126-132 .) is a template, wcag-10-F and 10-gmd-R are primers to amplify bidirectional promoter gal1,10. Using the total DNA of Saccharomyces cere...

Embodiment 3

[0043] Example 3. Construction of a genetically engineered strain of recombinant Saccharomyces cerevisiae producing 2'-fucosyllactose

[0044] Using human and Helicobacter pylori-derived α-1,2-fucosyltransferase as a template, new strains of Helicobacter pylori, Acetobacter sp., Bacillus cereus, Bacteroides unifomis, Bacteroides eggerthii, Neocallimastix californiae were discovered by blast analysis α-1,2-fucosyltransferase, respectively named Futhp (amino acid sequence is shown in SEQ ID No. 7, nucleotide sequence is shown in SEQ ID No. 8), Futas (amino acid sequence is shown in SEQ ID No. 9, the nucleotide sequence is shown in SEQ ID No. 10), Futbc (the amino acid sequence is shown in SEQ ID No. 11, the nucleotide sequence is shown in SEQ ID No. 12), Futbu (amino acid The sequence is shown in SEQ ID No. 15, the nucleotide sequence is shown in SEQ ID No. 16), Futbe (the amino acid sequence is shown in SEQ ID No. 13, and the nucleotide sequence is shown in SEQ ID No. 14) , Futnc...

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Abstract

The invention discloses a saccharomyces cerevisiae engineering strain with high yield of 2'-fucosyllactose. Through metabolic engineering modification, the saccharomyces cerevisiae can absorb lactoseand takes GDP-mannose as a precursor in a cell to synthesize GDP-fucose, alpha-1,2-fucosyltransferase derived from bacillus cereus is subjected to heterologous expression, the obtained recombinant strain can produce the 2'-fucosyllactose at a high yield, and an important reference is provided for industrial production of the 2'-fucosyllactose.

Description

Technical field [0001] The present invention belongs to the field of genetic engineering, and particularly relates to a recombinant Saccharomyces cerevisiae with high production of fucosyllactose, especially 2'-fucosyllactose and its application. Background technique [0002] 2'-Fucosyllactose (2-FL), as an important part of breast milk oligosaccharides, can promote the growth of beneficial microorganisms such as bifidobacteria in the human intestine, reduce the risk of pathogen infection in the intestine, and regulate immunity answer. Currently 2-FL has been used as an additive in infant formula milk powder. The large-scale synthesis of 2-FL has good economic benefits. Toxic reagents need to be added during the chemical synthesis of 2-FL, which is difficult to apply to the food industry. [0003] The synthesis of 2-FL in vivo is an effective method. The biosynthesis process of 2-FL is relatively clear: a molecule of donor GDP-fucose is combined with acceptor lactose under the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N9/12C12N9/88C12N9/04C12N9/10C12P19/12C12R1/865
CPCC12N9/1205C12N9/88C12N9/0006C12N9/1051C12P19/12C12Y402/01047C12Y101/01271C12Y204/01069
Inventor 孟祥锋刘巍峰徐明远
Owner SHANDONG UNIV
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