Saccharomyces cerevisiae S8-H with high yield of xylanase and application

A technology of Saccharomyces cerevisiae and Saccharomyces cerevisiae strains, applied in the field of microbial engineering, can solve the problems of lack of high-yielding strains and the like

Active Publication Date: 2018-12-07
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main limiting factor that restricts the production and application of xylanase in my country is the lack of high-yield strains that can adapt to large-scale production. Therefore, it is imminent to develop new high-yield xylanase strains with my country's independent intellectual property rights.

Method used

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  • Saccharomyces cerevisiae S8-H with high yield of xylanase and application
  • Saccharomyces cerevisiae S8-H with high yield of xylanase and application
  • Saccharomyces cerevisiae S8-H with high yield of xylanase and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Overlap extension PCR method to construct PαXC expression cassette

[0027] Aspergillus niger CICC2462 strain (China Industrial Microorganism Culture Collection Center) was subjected to PDA (composition: 200g potato boiled liquid, 20g glucose, 1.5g MgSO 4 ·7H 2 O, 1g KH 2 PO 4 , 20g Agar, deionized water constant volume to 1L) after plate activation, prepare seed liquid, seed liquid is inoculated in liquid fermentation medium (composition is: CaCl 2 5g, KH 2 PO 4 26g, FeSO 4· 7H 2 O 0.16g, yeast extract 10g, bran 20g, MgSO 4· 7H 2 O 10g, Tween-80 5ml, pH 5.0, distilled water 1000mL) 30°C, 150rpm, cultured for 72h, collected bacteria, and extracted RNA with AXYGEN total RNA extraction kit. Use the Taraka one-step reverse transcription kit to synthesize cDNA to amplify the xylanase xynB gene, the upstream and downstream primers xynB-F and xynB-R nucleotide coding sequences such as SEQ ID NO: 1 and SEQ ID NO: 2, Using S.Cerevisiae INVSc1 (purchased fr...

Embodiment 2

[0032] Example 2 Construction of S.Cerevisiae INVSc1-pYES2-PαXC-rDNA engineering bacteria

[0033] Using S.Cerevisiae INVSc1 genomic DNA as a template, amplify the core sequence of 2300bp (Genebank accession number BK006945.2) in the rDNA unit, the nucleotide sequence is as SEQ ID NO: 10, the upstream and downstream primers rDNA-F and rDNA-R core Nucleotide coding sequences such as SEQ ID NO: 11 and SEQ ID NO: 12, pYES2-PαXC and pMD19-T-rDNA were single-digested with SnaBI respectively, the target fragments were recovered, ligated with T4 ligase, and sequenced to obtain pYES2-PαXC and pMD19-T-rDNA. PαXC-rDNA expression vector such as image 3 shown. After the pYES2-PαXC-rDNA vector was linearized by SphI at the rDNA position, the LiAc / ssDNA method was used to transform the competent S. Cerevisiae INVSc1, and the transformed system was spread on the auxotrophic SC-U medium without uracil ( Purchased from Japan Clonetech Company), xylanase activity screening medium adopts xyla...

Embodiment 3

[0034] Example 3 Microdrop digital PCR identification of transformant copy number

[0035] For the obtained positive transformants, inoculate in YPD medium containing 20g / L peptone, 10g / L yeast powder and 10g / L glucose, the culture condition is 30°C, collect the bacteria after 48 hours of culture, and use Omega yeast genome extraction reagent The cassette extracts DNA, using the DNA as a template, and the ATC1 gene as an internal reference gene sequence (885bp, Genebank accession number CP020126.1) nucleotide coding sequence such as SEQ ID NO:13. XynB gene copy number identification using BIO-RAD QX200 digital droplet PCR. The nucleotide coding sequences of ATC1 upstream and downstream primers ATC1-F1 and ATC1-R1 are as SEQ ID NO: 14 and SEQ ID NO: 15, probe: 5'-HEX-CATCTTCGTTAGCTTCATCCGACGCTA-BHQ-3'; upstream primer xynB-F2 And the nucleotide coding sequence of the downstream primer xynB-R2 such as SEQ ID NO: 16 and SEQ ID NO: 17, probe: 5'-FAM-CCTGGTCAACTTTGCCCAGTCTAACAA-BH...

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Abstract

The invention relates to a saccharomyces cerevisiae strain S8-H with high yield of xylanase. The saccharomyces cerevisiae strain S8-H has the beneficial effects that the xylanase xynB genes from aspergillus niger CICC2462 are used for constructing expression boxes, utilizing a rDNA integration method to construct and obtain 8 copied xylanase gene strains, the expression quantity of the xylanase is325U / mL, 8 copied-xylanase engineered saccharomyces cerevisiaes with over-expressed HACl are obtained by transforming pYES6-PGK-HACl fragments to the strain, the expression quantity of the xylanase reaches 381U / mL, and by over-expressed HACl, the expression level of endoplasmic reticulum protein folding associated genes is improved; simultaneously, the invention provides a model for secretory expression of multiple copied exogenous genes by the saccharomyces cerevisiae, and the model can be utilized for constructing and expressing other genes; the saccharomyces cerevisiae strain with high yield of the xylanase can be advantageously applied in straw degration and the feed manufacturing process.

Description

technical field [0001] The invention belongs to the technical field of microbial engineering, and in particular relates to a high-yield xylanase Saccharomyces cerevisiae strain S8-H and an application thereof. Background technique [0002] Xylanase (xylanase) is a hydrolase that can degrade xylan into xylooligosaccharides and xylose. It not only plays a huge role in the energy industry, but also has a wide range of applications in the fields of feed, paper, food, etc. prospect. The main limiting factor restricting the production and application of xylanase in my country is the lack of high-yielding strains that can adapt to large-scale production. Therefore, it is imminent to develop new high-yielding xylanase strains with my country's independent intellectual property rights. [0003] Saccharomyces cerevisiae is a safe strain identified by the US Food and Drug Administration (FDA), and is an ideal host for the expression of food and feed enzyme preparations. Saccharomyces...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P19/00C12R1/865
CPCC12N9/248C12N15/81C12P19/00
Inventor 张斯童陈光孙旸王刚陈欢
Owner JILIN AGRICULTURAL UNIV
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