Penicillium decumbens engineered strain containing over-expressed xylanase and application thereof

A technology of Penicillium decumbens and xylanase, applied in the field of microbial engineering

Inactive Publication Date: 2012-05-30
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But its xylanase output still can't satisfy the demand of present industrial production well in the existing production method, therefore need to construct a strain of Penicillium decumbens (Penicillium decumbens) engineered bacterium that can overexpress xylanase so that reduce The production cost of xylanase, however, the search results have not yet seen the report of related work

Method used

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  • Penicillium decumbens engineered strain containing over-expressed xylanase and application thereof
  • Penicillium decumbens engineered strain containing over-expressed xylanase and application thereof
  • Penicillium decumbens engineered strain containing over-expressed xylanase and application thereof

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Embodiment 1

[0024] Example 1. Cloning and sequence determination of Penicillium decumbens xylanase I gene

[0025] First, the conserved region of the xylanase I gene (xyl1) of Penicillium decumbens was amplified and sequenced, and a 503bp DNA fragment of the conserved region was amplified. The specific process is as follows:

[0026] According to the amino acid conservation regions of some filamentous fungal xylanases (such as Aspergillus fumigatus Af293XynG1, Trichoderma.reesei xyn1, Trichoderma sp.SY xylanase, Trichoderma viride strainYNUCC0183endo-1, 4-beta-xylanase, Penicillium citrinum xyl1) submitted on NCBI Sequence design degenerate primer pair xyl1FA and xyl1RC, based on Penicillium decumbens (Penicillium decumbens) 114-2 (Qu Yinbo, Gao Peiji, Wang Zunong, "Selection of Cellulase Resistant Degradant Repression Mutants of Penicillium", Fungus Journal, 1984 04) chromosome as template for PCR amplification. PCR reaction system: 10×buffer (containing Mg 2+) 2.0 μl; dNTPs (10 mmol / ...

Embodiment 2

[0052] Embodiment 2. Construction and verification of recombinant plasmid pXXT-hph

[0053] The vector pXXT-hph containing hygromycin resistance gene (hph) expression cassette and Penicillium decumbens xyl1 cDNA gene expression cassette was constructed. Among them, the hph expression cassette comes from the pSilent-1 plasmid (Nakayashiki H, et al. (2005). RNA silencing as a tool for exploring gene function in ascomycete fungi. Fungal GenetBiol. 42 (4): 275-83), recumbent green Mold (Penicillium decumbens) xyl1 cDNA is subject to the regulation of TtrpC promoter and TtrpC in Trichoderma reesei QM9414, the specific construction process of this vector ( figure 1 )as follows:

[0054] Primers xyn2ps and xyn2pa were designed according to the GenBank sequence number (AY263380.1) of the promoter region of Trichoderma reesei QM9414xyn2, and the chromosomal DNA of T. reesei QM9414 was used as a template to amplify the promoter region of 1081 bp with pfu DNA polymerase. PCR reaction ...

Embodiment 3

[0062] Construction and molecular verification of embodiment 3.pXXT-hph vector transduction Penicillium decumbens transformant

[0063]Transform 10 μg of pXXT-hph plasmid into Penicillium decumbens JU-A10 (JU-A10 strain construction refers to Qu Y, Zhao X, GaO P.Appl Biochem Biotechnol, 1991, 28 / 29: 363-368), transform The process adopts the conventional protoplast transformation method, and the method is introduced with reference to Taina Karhunen, et al. (1993). High frequency one-stepgene replacement in Trichoderma reesei. I. Endoglucanase I overproduction. Mol Gen Genet, 241:515-522. The transformants were screened on the selection medium containing hygromycin resistance through primary screening, secondary screening and single spore isolation, and finally obtained 8 strains of Penicillium decumbens X series transformants with stable inheritance. After PCR (results see image 3 ) and Southern validation (results see Figure 4 ), proving that the above transformants were ...

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Abstract

The invention discloses a penicillium decumbens engineered strain containing over-expressed xylanase, which contains over-expressed penicillium decumbens xylanase I genes, is named penicillium decumbens X8 and is preserved in China Centre for Type Culture Collection on May 7, 2010 with a preservation number of CCTCC M 2010110. The invention also discloses the application of the strain in production of xylanase. The highest activity of the xylanase of the engineered strain of the invention reaches 219.82 IU / ml and is 1.57 times as high as that of an original host strain, which indicates that the strain is widely applied to the production of the xylanase.

Description

technical field [0001] The invention belongs to the field of microbial engineering; specifically, the invention relates to a strain of Penicillium decumbens engineering bacteria overexpressing xylanase I, and also relates to the application of the strain in producing xylanase. Background technique [0002] Xylanase refers to a group of enzymes that can specifically degrade hemicellulose xylan into xylooligosaccharides and xylose. It has shown wide application prospects in textile, papermaking, feed and food industries, especially in the pulp bleaching process, the application of xylanase can reduce the amount of chlorine-containing compounds, effectively reduce environmental pollution, and solve serious problems in papermaking The pollution problem of xylanase industry has extremely great practical significance (Gibbs MD, et al. (1995). Cloning, sequencing, and expression of a xylanase gene from the extreme thermophile Dictyoglomus thermophilumRt46B.1 and activity of the enz...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/15C12N9/42C12R1/80
Inventor 汪天虹杜春梅钟耀华李忠海张纪伟
Owner SHANDONG UNIV
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