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Penicillium oxalicum strain for increasing enzyme activities of cellulase and hemicellulase

A hemicellulase enzyme, Penicillium oxalicum technology, applied in the field of genetic engineering, can solve the problems of genetic modification without high-yielding cellulase strains, cell function differences, delayed expression, etc., and achieve good industrial development and application prospects and production capacity The effect of strengthening and shortening the production cycle

Active Publication Date: 2014-07-09
SHANDONG UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

In other filamentous fungi, some transcription factors that significantly regulate the expression of cellulase and hemicellulase genes have been found, such as the knockout of the gene encoding transcription factor CRE1 in Trichoderma reesei (Kubicek, C.P et al.2011.The CRE1carbon catabolite repressor of the fungus Trichoderma reesei: a master regulator of carbon assimilation. BMC Genomics, 12.), overexpression of Neurospora crassa transcription factor ClrB (Coradetti, S.T.et al.2013.Analysis of a conserved cellulase transcriptional regulator reveals inducer-independent production of cellulolytic enzymes in Neurospora crassa.Microbiologyopen.) can promote the high expression of cellulase and hemicellulase, however, there is no literature report on the overexpression of ClrB and the knockout of CRE1 in filamentous fungi Strong synergistic positive regulation of cellulase and hemicellulase expression
[0004] β-glucosidase is ubiquitous in cellulase-producing filamentous fungi (Chen, M., et al. 2013. Promotion of extracellular lignocellulolytic enzymes production by restraining the intracellular beta-glucosidase in Penicillium decumbens. Bioresour Technol, 137,33 -40.), but there are obvious differences in the cellular functions of β-glucosidase homologous proteins in different genera and species, such as the individual knockout strains of multiple β-glucosidase genes in Neurospora crassa to cellulase There was no obvious effect on the expression of (Znameroski,E.A et al.2013.Evidence for transceptor function of cellodextrin transporters in Neurospora crassa.J Biol Chem), but the homologous β-glucosidase encoding gene was knocked out in Trichoderma reesei cel1a (bglII), Δcel1a mutant strain under cellulose conditions, cellulase expression was significantly decreased and delayed expression (Zhou,Q et al.2012.Differential involvement of beta-glucosidases from Hypocrea jecorina in rapid induction of cellulase genes by cellulose and cellobiose.Eukaryot Cell)
However, according to searches, there is currently no report in the literature that the deletion of β-glucosidase Bgl2 can significantly promote the positive regulation of the transcription factor ClrB on the expression of cellulase and hemicellulase in filamentous fungi, let alone apply this to high-yielding fungi. Genetic Modification of Cellulase Strains

Method used

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  • Penicillium oxalicum strain for increasing enzyme activities of cellulase and hemicellulase
  • Penicillium oxalicum strain for increasing enzyme activities of cellulase and hemicellulase
  • Penicillium oxalicum strain for increasing enzyme activities of cellulase and hemicellulase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1, the construction of Penicillium oxalicum ClrB gene overexpression strain

[0069] (1) Cloning of Penicillium oxalicum ClrB coding region and terminal subregion

[0070] Using the extracted Penicillium oxalicum genomic DNA as a template, specific primers clrB-Fa and clrB-Ra were designed in the ClrB coding region and terminal subregion for PCR amplification to obtain Fragment 1. The primer sequences are as follows:

[0071] upstream primer

[0072] clrB-Fa:

[0073] 5'-AACAGCTACCCCGCTTGAGCAGACATCACCATGTTCCACACCTTTGAAGG-3'

[0074] downstream primer

[0075] clrB-Ra:

[0076] 5'-CCAATGGGATCCCGTAATCAATTGCCCGTAGCACCAGCGAAACATAC-3'.

[0077] (2) Penicillium oxalicum ClrB overexpression cassette promoter region gpdA (p) clone

[0078] The ClrB overexpression box promoter adopts the promoter sequence derived from the gene encoding 3'-phosphoglyceraldehyde dehydrogenase (gpdA) of Aspergillus nidulans. Amplification of the gpdA promoter sequence in a plasmi...

Embodiment 2

[0104] Embodiment 2, knockout of Penicillium oxalicum CreA coding gene

[0105] (1) Cloning of the upstream homology arm fragment of Penicillium oxalicum ⊿creA::bar knockout cassette

[0106] Using the extracted Penicillium oxalicum genomic DNA as a template, design specific primers CreA-F2 and CreAbar-R for PCR amplification of fragment 1 of the upstream homology arm of the CreA-encoded gene knockout cassette. The primer sequences are as follows:

[0107] Upstream primer CreA-F1:5'-CATTCCAGAGATGAACGACC-3'

[0108] downstream primer

[0109] CreAbar-R: 5'-GAAGGCCTGTAAGCGAATTAGCAAGCGTCGATGTGGGAACACCGGATA T-3'.

[0110] (2) Cloning of the homology arm fragment downstream of the Penicillium oxalicum ⊿creA::bar knockout cassette

[0111] Using the Genomic DNA of Penicillium oxalicum extracted by the method in the above general description as a template, design specific primers CreAbar-F and CreA-R2 for PCR amplification of the downstream homology arm fragment 2 of the knockout ...

Embodiment 3

[0130] Embodiment 3, knockout of Penicillium oxalicum β-glucosidase Bgl2 coding gene

[0131] (1) Cloning of the homology arm fragment upstream of the Penicillium oxalicum ⊿bgl2::hph knockout cassette

[0132] Using the extracted Penicillium oxalicum genomic DNA as a template, design specific primers bgl2-F2 and bgl2Hph-R for PCR amplification of fragment 1 of the upstream homology arm of the Bgl2 coding gene knockout cassette. The primer sequences are as follows:

[0133] Upstream primer Bgl2-F1:5'-GGCGGATACCCACTATGACC-3'

[0134] Downstream primer Bgl2hph-R:

[0135] 5'-GCTCCTTCAATATCAGTTAACGTCGTGGGTGTTGGTGGCAGAGTG-3'.

[0136] (2) Cloning of the downstream homology arm fragment of Penicillium oxalicum Bgl2 knockout cassette

[0137] Using the extracted Penicillium oxalicum genomic DNA as a template, design specific primers bgl2Hph-F and bgl2-R2, and PCR amplify the downstream homology arm fragment 2 of the Bgl2 coding gene knockout cassette. The primer sequences are as f...

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Abstract

The invention discloses a penicillium oxalicum strain for increasing enzyme activities of cellulase and hemicellulase. The strain is named penicillium oxalicum RE-10, and is collected in the China General Microbiological Culture Collection Center with the collection number CGMCC No. 8922 on March 17, 2014. The invention further discloses an application of the strain to production of cellulase and hemicellulase. As proved by experiments, the filter paper activity is increased by 52 times in comparison to that of an original strain by using the strain disclosed by the invention under the condition of an enzyme production culture medium. Compared with the conventional existing industrial stain, the engineering strain disclosed by the invention is remarkably shorter in the production period, higher in the yields of cellulase and hemicellulase and lower in production cost, can be applied to large-scale production of cellulase and hemicellulase, and has good industrial development and application prospects.

Description

technical field [0001] The invention relates to an engineering bacterial strain with cellulase and hemicellulase activity, in particular to a Penicillium oxalicum bacterium which improves the activity of cellulase and hemicellulase, and belongs to the field of genetic engineering. Background technique [0002] Penicillium oxalicum is a filamentous fungus with high cellulase and hemicellulase production (Qu, et al. 2013. Improving lignocellulolytic enzyme production with Penicillium: from strain screening to systems biology. Biofuels, 4(5), 12) , has made extensive progress in the expression regulation of cellulase and the genetic modification of high-yield cellulase, and this strain has been applied to the industrial production of cellulase. With the depletion of petrochemical energy resources in nature, renewable energy is getting more and more attention, especially lignocellulose, which is the most abundant renewable resource in nature, and its related biorefinery industry...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/80C12N9/42C12R1/80
Inventor 曲音波李忠海姚光山秦玉琪李雪芝
Owner SHANDONG UNIV
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