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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AI Technical Summary

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, 1

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

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0068] Example 1. Construction of ClrB gene overexpression strain of Penicillium oxalicum

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

[0070] Using the extracted Penicillium oxalicum genomic DNA as a template, specific primers clrB-Fa and clrB-Ra were designed in the coding region and terminal region of ClrB for PCR amplification to obtain fragment 1. The primer sequence is 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 of

[0078] The ClrB overexpression cassette promoter uses a promoter sequence derived from the 3'-glyceraldehyde phosphate dehydrogenase encoding gene (gpdA) of Aspergillus nidulans. Amplification of gpdA promoter sequence with plasmid P AN7-1 w...

Example Embodiment

[0104] Example 2. Knockout of CreA encoding gene of Penicillium oxalicum

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

[0106] Using the extracted Penicillium oxalicum genomic DNA as a template, specific primers CreA-F2 and CreAbar-R were designed to PCR amplify the upstream homology arm segment 1 of the CreA-encoding gene knockout box. 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 homology arm fragments downstream of Penicillium oxalicum ⊿creA::bar knockout box

[0111] Using the Penicillium oxalicum genomic DNA extracted by the above general instructions as a template, specific primers CreAbar-F and CreA-R2 were designed to PCR amplify the downstream homology arm segment 2 of the CreA-encoding gene knockout box. The primer sequences are as follows: ...

Example Embodiment

[0130] Example 3 Knockout of the gene encoding Penicillium oxalicum β-glucosidase Bgl2

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

[0132] Using the extracted Penicillium oxalicum genomic DNA as a template, specific primers bgl2-F2 and bgl2Hph-R were designed to PCR amplify the upstream homology arm segment 1 of the knockout box encoding the Bgl2 gene. 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 segment of the Penicillium oxalicum Bgl2 knockout box

[0137] Using the extracted Penicillium oxalicum genomic DNA as a template, specific primers bgl2Hph-F and bgl2-R2 were designed to amplify the downstream homology arm segment 2 of the knockout box encoding the Bgl2 gene by PCR. The primer sequences are as follows:

[0138] Upstr...

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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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