Construction method of recombinant Yarrowia lipolytica for synthesis of xylitol and strain thereof

A technology of Yarrowia lipolytica and yeast strains, which is applied in the field of fermentative synthesis of xylitol, construction method of recombinant Yarrowia lipolytica and its strains, which can solve the problems of increasing synthesis steps and consuming microbial cell resources

Active Publication Date: 2020-03-13
SHANGHAI JIAO TONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The whole process needs to go through the intermediate of D-arabitol, which

Method used

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  • Construction method of recombinant Yarrowia lipolytica for synthesis of xylitol and strain thereof
  • Construction method of recombinant Yarrowia lipolytica for synthesis of xylitol and strain thereof
  • Construction method of recombinant Yarrowia lipolytica for synthesis of xylitol and strain thereof

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Experimental program
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Effect test

Embodiment 1

[0097] Embodiment 1, the acquisition of Yarrowia lipolytica ery929 (CGMCC 18478 bacterial strain)

[0098] Take 5 grams of fresh bee hives from different sources each, cut each portion into small pieces with a length less than 5 mm with sterilized scissors, soak in 20 ml of sterile water containing 0.05% Tween 40, stir for 1 hour, and centrifuge at 5000 rpm For 10 minutes, discard the supernatant and honeycomb fragments, suspend the precipitate with 1 ml of sterile water, and spread every 200 microliters in sterilized hypertonic solid medium (ingredients: anhydrous glucose 400g / L, yeast powder 12g / L , ammonium citrate 5g / L, peptone 3g / L, agar 15g / L, pH5.5), cultured at 30°C for 7 days. The yeast-like colonies were selected for pure culture, and the pure cultured yeast colonies were taken to ferment and synthesize erythritol one by one. The composition of the liquid medium is: anhydrous glucose 300g / L, yeast powder 8g / L, ammonium citrate 5g / L, peptone 3g / L, copper chloride 0...

Embodiment 2

[0112] Embodiment 2, constructing the recombinant Yarrowia lipolytica strain capable of synthesizing xylitol

[0113] (1) Overexpression of xylitol dehydrogenase gene in Yarrowia lipolytica.

[0114] The xylitol dehydrogenase gene (Candida maltosa, SEQ ID NO 9) of the optimized synthetic Candida maltosa was connected to the integrated expression plasmid vector pSWV-Int (schematic figure 2 ). The vector is based on the pUC series of common cloning vectors, adding common DNA element sequences, such as 26S rDNA left and right homology arm sequences, artificially synthesized promoter hp4d sequence, transcription elongation factor gene terminator TT TEFSequence, invertase selection marker gene sequence Suc2, Escherichia coli plasmid replication origin sequence ori, ampicillin resistance gene sequence DNA elements, these basic DNA elements those skilled in the art can be found from NCBI database (https: / / www .ncbi.nlm.nih.gov / ). The schematic diagram of the integrated expressio...

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Abstract

The invention discloses a construction method of recombinant Yarrowia lipolytica for synthesis of xylitol and a strain thereof. Yarrowia lipolytica is used as a synthetic chassis. Through the improvement means of metabolic engineering, the yeast undergoes gene splicing, genes for synthesizing xylitol from glucose, fructose, glycerol and starch as carbon sources are introduced, and metabolic pathways of synthesis by-products are blocked, so that recombinant Yarrowia lipolytica can synthesize xylitol from glucose, fructose, glycerol and starch as carbon sources by fermentation, and an engineering strain for synthesizing xylitol from carbon sources such as glucose and the like is obtained. After fermentation, xylitol crystals are obtained by separation of a bacterial liquid, ion exchange of aclarified fermentation liquid, decolorization, concentration, crystallization and other processes. The construction method of the engineering yeast for synthesizing xylitol from carbon sources such as glucose and the Yarrowia lipolytica strain obtained by the method and for synthesizing xylitol from carbon sources such as glucose can simplify existing methods for chemically synthesizing xylitol and have better application value.

Description

technical field [0001] The invention belongs to the field of food biotechnology, and relates to a method for constructing recombinant Yarrowia lipolytica for synthesizing xylitol and strains thereof; more specifically, it relates to using Yarrowia lipolytica as a chassis microorganism through metabolic engineering, genetic engineering and Synthetic biology means to construct a method for fermentatively synthesizing xylitol, using this method to obtain a recombinant Yarrowia lipolytica that can ferment and synthesize xylitol from glucose and other carbon sources, and using the recombinant strain to ferment and synthesize xylitol Methods. Background technique [0002] Xylitol (xylitol) is a pentahydric alcohol with a CAS number of 87-99-0 and a molecular weight of 152.15 Daltons. It is a common food additive and is often used in the preparation of chewing gum, dairy products, candies and other foods. Reducing the use of sucrose has good effects on preventing oral diseases, re...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/53C12N15/55C12N15/54C12N15/31C12N15/61C12P7/18C12R1/645
CPCC12N9/0006C12N9/16C12N9/0036C12N9/1022C12N9/12C12N9/90C07K14/39C12P7/18C12Y101/01255C12Y202/01001C12N2800/22C12Y101/01307C12Y101/01009C12Y106/01002C12N15/52C12N15/815C12R2001/645C12N1/16C12N2800/102
Inventor 程海荣
Owner SHANGHAI JIAO TONG UNIV
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