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Method for efficiently synthesizing glucan by constructing recombinant saccharymyces cerevisiae

A technology of Saccharomyces cerevisiae and glucan, applied in the field of biomedicine, can solve the problems of low yield, time-consuming screening, and insufficient effect on the yield of β-D-glucan, and achieve the effect of increasing yield and content

Active Publication Date: 2018-02-16
RAYTING BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These two methods are not effective enough to increase the yield of β-D-glucan, especially the method of medium optimization, the increased yield is very low
In addition, the method of mutation breeding needs a lot of time to screen useful strains, and has a certain degree of blindness.

Method used

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  • Method for efficiently synthesizing glucan by constructing recombinant saccharymyces cerevisiae
  • Method for efficiently synthesizing glucan by constructing recombinant saccharymyces cerevisiae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Construction of embodiment 1 recombinant strain

[0033] Using the episomal vector YEp series vector YEp351 as the carrier, connect the FKS1 gene with a sequence such as Gene ID 851055 to the YEp351 as the carrier to construct the YEp351-FKS1 recombinant vector, and then transform it into Saccharomyces cerevisiae CEN.PK2-1C to obtain a transformant. Access the corresponding defect type plate. The colony PCR was verified, and the recombinant strains correctly transferred to the target fragment were obtained.

[0034] Recombinant strains expressing other genes were constructed in a similar manner.

Embodiment 2

[0035] Example 2 Extraction of Yeast Cell Wall Glucan

[0036] After the cells were collected, they were washed by centrifugation. Freeze-dried and ground to make dry yeast powder. Dry yeast powder was formulated with pH 6.0 phosphate buffer solution to make 80g / L bacterial suspension, and papain and neutral protease were added to hydrolyze at 60°C for 1 hour. Then it was extracted with NaOH solution at 51°C for 1h.

Embodiment 3

[0037] Embodiment 3 Determination of Dextran Content

[0038] Weigh 100mg of extracted crude dextran, add 2M H 2 SO 4 5ml of the solution was hydrolyzed at 96°C for 6h. The pH was adjusted to neutral, and the glucose concentration was measured by the DNS method.

[0039] Such as figure 1 Shown is the effect of the expression of a single key gene in the β-D-glucan synthesis pathway on the β-D-glucan content in the cell wall of Saccharomyces cerevisiae, where the control specifically refers to the unmodified original strain. From figure 1 It can be seen that the recombinant bacteria expressing FKS1, GSC2, RHO1, KRE5, KRE6 genes, compared with the control, the content of β-D-glucan increased by 67%, 44%, 56%, 18%, 27%, respectively. %.

[0040] Such as figure 2 As shown, the co-expression of multiple key genes in the β-D-glucan synthesis pathway affects the β-D-glucan content in the cell wall of Saccharomyces cerevisiae, where the control specifically refers to the origin...

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Abstract

The invention discloses a method for efficiently synthesizing glucan by constructing recombinant saccharymyces cerevisiae, and belongs to the field of biomedicines. Saccharymyces cerevisiae strains capable of producing a high yield of beta-D-glucan are obtained by regulating and controlling expression quantities of glucan synthase and relevant genes and the substrate level. According to recombinant bacteria, by expression of genes such as FKS1, GSC2, RHO1, KRE5 and KRE6, the content of beta-D-glucan is obviously increased, wherein the yield of beta-D-glucan of the recombinant bacteria expressing the FKS1 and the RHO1 is respectively increased by 67 percent and 55 percent, and the yield of beta-D-glucan of the recombinant bacteria expressing the FKS1, GSC2 and RHO1 is increased by 299 percent.

Description

technical field [0001] The invention relates to a method for constructing recombinant Saccharomyces cerevisiae to efficiently synthesize glucan, belonging to the field of biomedicine. Background technique [0002] 85-90% of the yeast cell wall is polysaccharide, 10-13% is protein, and also contains a small amount of fat and other substances. The components of yeast cell wall polysaccharides are composed of water-soluble mannose, alkali-soluble and alkali-insoluble β-D-glucan, and a small amount of chitin. Yeast β-D-glucan is a mixed polysaccharide composed mainly of β-1,3-glucan and supplemented by β-1,6-glucan. [0003] Yeast β-D-glucan can specifically combine with animal and human immune cells, including monocytes, macrophages, neutrophils and natural killer cells, and enhance the immune function of host cells. In addition, yeast β-D-glucan also has the effects of anti-tumor, enhancing the body's anti-radiation, lowering cholesterol and blood lipids, etc. [0004] At p...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12P19/08A23L33/14A61K36/06A61P35/00A61P39/00A61P3/06A61P17/16C12R1/865
CPCA23V2002/00A61K36/06A23L33/14C12P19/08C12N1/185C12R2001/865A23V2200/308A23V2200/3262
Inventor 康振杜养标陈坚周星
Owner RAYTING BIOTECHNOLOGY CO LTD