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Optimized saccharomyces cerevisiae strain capable of producing fucosyllactose at high yield and application of saccharomyces cerevisiae strain

A technology of fucosyllactose and Saccharomyces cerevisiae, which is applied in the field of genetic engineering, can solve the problems of pollution, difficulty in meeting industrial production, 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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  • Optimized saccharomyces cerevisiae strain capable of producing fucosyllactose at high yield and application of saccharomyces cerevisiae strain
  • Optimized saccharomyces cerevisiae strain capable of producing fucosyllactose at high yield and application of saccharomyces cerevisiae strain
  • Optimized saccharomyces cerevisiae strain capable of producing fucosyllactose at high yield and application of saccharomyces cerevisiae strain

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1. Construction of recombinant Saccharomyces cerevisiae genetically engineered strains capable of absorbing lactose

[0035] In the construction of the recombinant engineering strain in this example, Saccharomyces cerevisiae W303-1a was used as the starting strain.

[0036] Utilize the Omega kit to extract the total DNA of Kluyveromyceslactis (Kluyveromyceslactis), and use primers Lac12-F and Lac12-R to amplify the sequence of lactose permease (Lactosepermease) therefrom, named Lac12, its amino acid sequence is as follows Shown in SEQ ID No.1, the nucleic acid sequence is shown in SEQ ID No.2.

[0037] Using Saccharomyces cerevisiae (Saccharomyces cerevisiae) W303-1a total DNA 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 Out of the cyc1 terminator, P was constructed by SOE-PCR gal1 -lac12-T cyc1 The expression cassette is connected to the pRS304 vector cut with BamHI ...

Embodiment 2

[0043] Example 2. Construction of recombinant Saccharomyces cerevisiae genetically engineered strains producing GDP-fucose

[0044] From Escherichia coli (Escherichia coli) K12, the sequence of GDP-mannose dehydratase (GDP-mannose-4,6-dehydratase) was amplified with primers GMD-F and GMD-R, named Gmd, and its amino acid sequence is as follows: Shown in SEQ ID No.3, its nucleotide sequence is shown in SEQ ID No.4. The sequence of GDP-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 nucleotide The sequence is shown in SEQ ID No.6.

[0045] Using pUMRI-A plasmid (Ye, L., X.Lv, and H.Yu, Assembly of biosynthetic pathways in Saccharomyces cerevisiae using a marker recyclable integrative plasmidtoolbox. Frontiers of Chemical Engineering in China, 2017.11(1): p.126-132 .) as a template, and wcag-10-F and 10-gmd-R as primers to amplify the bidirectional promoter gal1,10. Using ...

Embodiment 3

[0050] Example 3. Construction of recombinant Saccharomyces cerevisiae genetically engineered strains producing 2'-fucosyllactose

[0051] Using human and Helicobacter pylori-derived α-1,2-fucosyltransferases as templates, new α- 1,2-fucosyltransferase, respectively named Futhp (amino acid sequence as shown in SEQ ID No.7, nucleotide sequence as shown in SEQ ID No.8), Futas (amino acid sequence as 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 (the amino acid sequence is shown in SEQ ID No.12), Futbu (the amino acid sequence is shown in Shown in SEQ ID No.15, nucleotide sequence is shown in SEQ ID No.16), Futbe (amino acid sequence is shown in SEQ ID No.13, nucleotide sequence is shown in SEQ ID No.14), Futnc (The amino acid sequence is shown in SEQ ID No. 17, and the nucleotide sequence is shown in SEQ ID No. 18).

[0052] The above α-...

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Abstract

The invention discloses an optimized saccharomyces cerevisiae engineering strain capable of highly producing 2'-fucosyllactose. Through modification by metabolic engineering, the saccharomyces cerevisiae can absorb lactose and takes GDP-mannose as a precursor in a cell to synthesize GDP-fucose, and alpha-1,2-fucosyltransferase derived from bacillus cereus is heterologously expressed. The 2'-fucosyllactose with a high yield can be obtained by the obtained recombinant strain, and the 2'-fucosyllactose yield can be increased to about 3.8 g / L through subsequent overexpression of the GDP-mannose synthesis related enzyme. Therefore, an important reference is provided for industrial production of the 2'-fucosyllactose from the saccharomyces cerevisiae.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a recombinant Saccharomyces cerevisiae strain with high yield of fucosyllactose, especially high yield of 2'-fucosyllactose and its application. Background technique [0002] 2'-fucosyllactose (2-FL), as an important component of breast milk oligosaccharides, can promote the growth of beneficial microorganisms such as bifidobacteria in the human intestinal tract, reduce the risk of pathogenic infection in the intestinal tract, and regulate immunity answer. At present, 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 in the food industry. [0003] Synthesizing 2-FL in vivo is an effective method. The biosynthesis process of 2-FL has been relatively clear: a molecule of donor GDP-fucose is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N9/12C12N9/88C12N9/04C12N9/10C12N9/90C12P19/12C12R1/865
CPCC12N9/1205C12N9/88C12N9/0006C12N9/1051C12N9/90C12N9/1241C12P19/12C12Y402/01047C12Y101/01271C12Y204/01069C12Y504/02008C12Y503/01008C12Y207/01086C12Y207/07013
Inventor 刘巍峰孟祥锋徐明远
Owner SHANDONG UNIV
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