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Method for constructing recombinant yarrowia lipolytica for synthesizing erythritol and strain of yarrowia lipolytica

A technology of Yarrowia lipolytica and erythritol, which is applied in the field of food biology and can solve problems such as reduced synthetic performance

Active Publication Date: 2020-07-03
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is not arbitrarily overexpressing or knocking out a gene to obtain a strain with significantly improved performance. Although some genes are genes in the synthetic pathway, overexpression will lead to a decrease in synthetic performance.

Method used

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  • Method for constructing recombinant yarrowia lipolytica for synthesizing erythritol and strain of yarrowia lipolytica
  • Method for constructing recombinant yarrowia lipolytica for synthesizing erythritol and strain of yarrowia lipolytica
  • Method for constructing recombinant yarrowia lipolytica for synthesizing erythritol and strain of yarrowia lipolytica

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1. Overexpression of hexokinase gene in Yarrowia lipolytica

[0096] The hexokinase expression cassette was synthesized according to the following sequence: upstream 26S rDNA sequence → promoter (hp4d) → hexokinase gene sequence → hexokinase terminator sequence → loxP → promoter (TEF) → xylitol dehydrogenase gene sequence → Terminator sequence → loxP → downstream 26S rDNA sequence. The sequence of the synthetic expression cassette containing hexokinase is SEQ ID No.24.

[0097] The schematic diagram of the DNA elements in the integrated expression box constructed containing the hexokinase gene is as follows figure 2 As shown, the hexokinase gene can be derived from the hexokinase gene of Saccharomyces cerevisiae, Pichia stipitis, Binding yeast, Candida sphaeroides, or Yarrowia lipolytica itself. Here, Yarrowia lipolytica is selected Yeast's own hexokinase gene (SEQ ID No. 6). The selection marker can be replaced by an auxotrophic marker such as the ura3 gene, or ...

Embodiment 2

[0100] Example 2. Overexpression of transketolase gene in Yarrowia lipolytica

[0101] Replace the hexokinase gene in Example 1 with the transketolase gene. The rest of the DNA elements can remain unchanged or can be changed accordingly. For example, the promoter sequence can be other promoter sequences, such as the glycerol dehydrogenase gene promoter sequence. Wait. The selection marker xylitol dehydrogenase gene can also be a sucrose hydrolase gene. The sequence of the synthetic expression cassette containing the transketolase gene is SEQ ID No. 25.

[0102] The schematic diagram of the DNA element in the integrated expression box containing the transketolase gene is constructed as Figure 4 As shown, the transketolase gene comes from but is not limited to the following microorganisms: Saccharomyces cerevisiae, Candida magnolia, Pichia stipitis, or Yarrowia lipolytica itself. The transketolase gene TKL1 (SEQ ID No. 10) of Yarrowia lipolytica itself is used here. The methods ...

Embodiment 3

[0107] Example 3. Overexpression of transaldolase gene in Yarrowia lipolytica

[0108] The expression cassette containing the transaldolase gene was synthesized, and its sequence is SEQ ID No.26. The schematic diagram of the DNA element in the integrated expression box containing the transaldolase gene is constructed as Figure 5 Shown. The transaldolase gene comes from but is not limited to the following microorganisms: Pichia stipitis, Candida tropicalis, Komagataella phaffii yeast, Debaryomyces hansenii, Yarrowia lipolytica. The transaldolase gene TAL (SEQ ID No. 11) of Yarrowia lipolytica itself is used here. The methods of DNA transformation, transformant strain screening and selection marker recovery are the same as in Example 1. The chassis strain used can be the transformant ery::HK::TKL1 with overexpression of the hexokinase gene obtained in Example 2, or other strains that can synthesize erythritol (such as the patent previously obtained by the inventor) Strain Yarr...

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Abstract

The invention discloses a method for constructing recombinant yarrowia lipolytica for synthesizing erythritol and a strain of the yarrowia lipolytica. The yarrowia lipolytica is adopted as a syntheticchassis, the yeast is subjected to gene editing through a metabolic engineering improvement means, the capability of erythritol synthesizing is enhanced, and the route of synthesizing by-products isblocked. The yeast strain is yarrowia lipolytica ery949-4 delta CGMCC No. 19351. Glucose with the initial concentration of 50-350 g / L is adopted as a carbon source, and a nitrogen source with the initial concentration of 5-30g / L and inorganic salts are taken as raw materials, high-temperature sterilization and cooling are carried out to be incubated with the yarrowia lipolytica yeast strain, and continuous fermentation or batch supplemented fermentation is carried out under the aerobic conditions; and after fermentation is finished, erythritol is purified from the fermentation broth. The strain can efficiently synthesize erythritol by taking glucose and other carbon source as the raw materials, does not synthesize by-products (such as mannitol and the like), and does not consume erythritol.

Description

Technical field [0001] The present invention belongs to the field of food biotechnology, and relates to a method for constructing a recombinant Yarrowia lipolytica synthesizing erythritol and its strain; more specifically, it relates to a microorganism using Yarrowia lipolytica as a chassis through metabolic engineering and genetic engineering. Engineering means to construct a method for efficient fermentation and synthesis of erythritol, use this method to obtain recombinant Yarrowia lipolytica that can ferment to synthesize erythritol from carbon sources such as glucose, and use the recombinant strain to ferment and synthesize erythritol Sugar alcohol method. Background technique [0002] Erythritol is a naturally occurring tetrahydric alcohol. It is often used as an intracellular carbon source storage substance in seaweed and fungi. Erythritol is found in some fruits such as grapes, melons and pears. The existence of (Munro et al. Food Chem Toxicol. 1988; 36(12):1139-74.). A...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N1/19C12P7/18C12R1/645
CPCC12N15/815C12P7/18C12N9/1022C12N9/1205C12N9/1051C12Y207/01001C12Y202/01001C12Y202/01002
Inventor 程海荣王楠池萍
Owner SHANGHAI JIAO TONG UNIV
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