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Recombinant yeast strain for fermenting erythritol under high nitrogen condition, construction method and application thereof

A technology of yeast strain and construction method, which is applied in the field of DNA recombination, can solve the problems of fermentation sluggishness and low efficiency, and achieve the effects of promoting application, shortening fermentation time, and solving fermentation delay

Active Publication Date: 2019-08-16
HUAIYIN TEACHERS COLLEGE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In view of the deficiencies pointed out in the above-mentioned background technology, the present invention provides a recombinant yeast strain for fermenting erythritol under high nitrogen conditions and its construction method and application. By knocking out the SNF1 gene of the Snf1 protein, nitrogen starvation stress and erythritol stress are relieved The coupling between alcohol synthesis enables Yarrowia lipolytica to synthesize erythritol under high nitrogen conditions, which solves the problem of sluggish fermentation and low efficiency in erythritol synthesis initiated by nitrogen starvation in traditional fermentation

Method used

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  • Recombinant yeast strain for fermenting erythritol under high nitrogen condition, construction method and application thereof
  • Recombinant yeast strain for fermenting erythritol under high nitrogen condition, construction method and application thereof
  • Recombinant yeast strain for fermenting erythritol under high nitrogen condition, construction method and application thereof

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Embodiment 1

[0028] Construction method of recombinant yeast strain fermenting erythritol under a kind of high nitrogen condition

[0029] (1) SNF1 gene sequence analysis and cloning

[0030] Referring to SNF1 in the whole genome of Yarrowia lipolytica SWJ-1b (isolated from Bohai fish intestine), the gene with Genbank gene accession number XP_502312 is the SNF1 gene. The SNF1 gene sequence is shown in SEQ NO.1.

[0031] (2) Construction of gene SNF1 knockout strain

[0032] Gene fusion technology was used to construct the gene knockout module, and the Yarrowia lipolytica genome was used as a template to design amplification primers, and the upstream and downstream fragments of the SNF1 gene and the Yarrowia lipolytica screening marker URA3 fragment were respectively amplified by PCR reactions. Upstream and downstream fragment PCR reaction system: 2 μL yeast genomic DNA, 1 μL primers, 2 μL dNTP, 5 μL MgCl 2 , 5 μL LATaq polymerase buffer, and 0.5 μL LA Taq polymerase. PCR cycle conditio...

Embodiment 2

[0047] Fermentation of a recombinant yeast strain to produce erythritol under high nitrogen conditions

[0048] The original bacterial strain and the recombinant yeast strain obtained in Example 1 (the Yarrowia lipolytica strain whose SNF1 gene was knocked out) were respectively placed on the YPD culture plate (20.0g / L glucose, 20.0g / L peptone, 10.0g / L Yeast powder and 15.0g / L agar), cultured at 28°C for 3 days to grow a single colony;

[0049] Then inoculate a single colony in low-nitrogen and high-nitrogen fermentation media, and shake culture at 28°C until DO 600 =30, obtain the seed liquid, insert the seed liquid in the erythritol fermentation medium, the composition of the erythritol fermentation medium is: glycerol 100g / L, ammonium sulfate 12.5g / L, K 2 HPO 4 0.23g / L, MgSO 4 1.0g / L, yeast powder 1.0g / L, sodium chloride 25.0g / L, prepared with distilled water. The inoculum size was 10%, and cultured with shaking at 28°C for 5 days. The culture solution was centrifuge...

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Abstract

The invention discloses a recombinant yeast strain for fermenting erythritol under high nitrogen condition, a construction method and an application thereof. The recombinant strain is constructed by:obtaining the SNF1 gene in the whole genome of Yarrowia lipolytica, using the Yarrowia lipolytica genome as a template to separately amplify the upstream and downstream fragments of the SNF1 gene andthe URA3 fragment by PCR reaction, ligating the upstream and downstream fragments of the SNF1 gene to the upstream and downstream of the URA3 fragments by an overlap extension PCR method to obtain aSNF1 gene knockout component, transferring the SNF1 gene knockout module to the selected uracil-deficient Yarrowia lipolytica strain, and screening to obtain the Yarrowia lipolytica recombinant strainin which the SNF1 gene was knocked out. The strain eliminates the coupling between nitrogen hunger stress and erythritol synthesis by knocking out the SNF1 gene of Snf1 protein. The recombinant yeaststrain of the invention can synthesize erythritol under high nitrogen conditions, and the fermentation efficiency is obviously improved, which solves the problem of delayed fermentation and low yieldin industrial production of erythritol.

Description

technical field [0001] The invention belongs to the technical field of DNA recombination, and in particular relates to a recombinant yeast strain for fermenting erythritol under high nitrogen conditions, a construction method and application thereof. Background technique [0002] Erythritol is a commonly used non-sugar sweetener with extremely low calorie value (0.2 kcal / g) and zero-glycemic index. It is the only sugar that is directly excreted through urine without participating in human metabolism. Alcohol, so it is very suitable for people with diabetes, obesity and cardiovascular disease, and has become a very important special dietary additive. The main method of obtaining erythritol is the fermentation method. Under the environment of high osmotic pressure, Yarrowia lipolytica (Yarrowia lipolytica) can directly use various industrial and agricultural wastes to generate erythritol. and low-cost fermentation have unique advantages. Y.lipolytica et al. reported the synt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/18C12N15/81C12R1/645
CPCC12P7/18C12N15/81
Inventor 刘晓燕赵朴素宋洁王致鹏夏军许家兴王晓宇蒋叶涛
Owner HUAIYIN TEACHERS COLLEGE
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