Penicillium oxalicum host strain for enhancing expression of filamentous fungi protein

A technology of Penicillium oxalicum and filamentous fungus, applied in the field of genetic engineering, can solve the problems that cellulase cannot be induced and secreted, protein products are easy to degrade, low secretion background, etc., and achieve good industrial development and application prospects, production The target protein cycle is short and the effect of high protein expression

Inactive Publication Date: 2014-08-06
SHANDONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage is that some protein products are easy to degrade, the expression level is uncontrollable, and excessive glycosylation is common when expressing filamentous fungal proteins
[0004] At present, the main expression host of filamentous fungi is the mutant strain Δxyr of Trichoderma reesei, because of the lack of a major positive regulator of cellulase, cellulase cannot be induced and secreted extracellularly. Thereby, a low protein secretion background is formed when culturing on glucose, which is conducive to heterologous expression to obtain a relatively pure target protein, but Trichoderma reesei grows slowly, and it takes about 7 days to complete sporulation on the PDA m

Method used

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  • Penicillium oxalicum host strain for enhancing expression of filamentous fungi protein
  • Penicillium oxalicum host strain for enhancing expression of filamentous fungi protein
  • Penicillium oxalicum host strain for enhancing expression of filamentous fungi protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, the construction of Penicillium oxalicum PGA3 gene persistent activation strain

[0058] (1) Cloning of the fragment of the mutant coding region of Penicillium oxalicum pga3

[0059] Using pUC-Mpga3 as a template, design specific primers MG3-F: and MG3-R: in the mutated pga3 coding region for PCR amplification to obtain fragment 1, the primer sequences are as follows:

[0060] upstream primer

[0061] MG3-F:

[0062] 5'-CTCATCTTCATCGCATCCCAA-3'

[0063] downstream primer

[0064] MG3-R:

[0065] 5'-AATGGGATCCCGTAATCAATTGCCCTCGGGAGGAAGACAAGAAGG-3'.

[0066] (2) Cloning of the downstream homology arm of Penicillium oxalicum pga3

[0067] Using the extracted Penicillium oxalicum genomic DNA as a template, primers MG3-3F and MG3-3R were designed for PCR amplification to obtain Fragment 2. The primer sequences are as follows:

[0068] upstream primer

[0069] MG3-3F

[0070] 5'-CAAGAGCGGCTCATCGTCACCCCCATTCCTCTTCCGCACGATGATT-3'

[0071] Downstream prim...

Embodiment 2

[0095] Embodiment 2, knockout of Penicillium oxalicum amy15A promoter and coding gene

[0096] (1) Cloning of the upstream homology arm fragment of Penicillium oxalicum ⊿amy15A::bar knockout cassette

[0097] Using the extracted Penicillium oxalicum genomic DNA as a template, specific primers amy15A-F and amy15Abar-R were designed for PCR amplification of the upstream homology arm fragment 1 of the amy15A promoter. The primer sequences are as follows:

[0098] Upstream primer amy15A-F: 5'-CAGCATCCAATGGACGTTCA-3'

[0099] downstream primer

[0100] amy15Abar-R:5'-GCCTGTAAGCGAATTAGCAAGCGTCCAAAGTTTCCTTGGCAGCGT-3'.

[0101] (2) Cloning of the downstream homology arm fragment of Penicillium oxalicum ⊿amy15A::bar knockout cassette

[0102] Using the Genomic DNA of Penicillium oxalicum extracted by the method mentioned above as a template, design specific primers amy15Abar-F and amy15A-R for PCR amplification of the downstream homology arm fragment 2 of the amy15A coding gene knoc...

Embodiment 3

[0122] Example 3. Overexpression of Penicillium oxalicum exocellulase CBHI gene in Penicillium oxalicum M3QΔ15A

[0123] (1) Cloning of the upstream promoter fragment of Penicillium oxalicum amy15A(p)::cbh1::hph overexpression cassette

[0124] Using the extracted Penicillium oxalicum genomic DNA as a template, design specific primers P15A-F and P15ACBH-R for PCR amplification of the upstream promoter fragment 1 of the CBHI overexpression cassette. The primer sequences are as follows:

[0125] Upstream primer P15A-F: 5'-AGTCCCAGATCGCTTGTGACA-3'

[0126] Downstream primer P15ACBH-R:

[0127] 5'-GTAGGAGATGGAACCCTTCATCTTTGGTAGACAGTTGAAAGTGTCG-3'.

[0128] (2) Cloning of Penicillium oxalicum CBHI coding region and terminal subregion

[0129] Using the extracted Penicillium oxalicum genomic DNA as a template, specific primers CBHI-F and CBHI-R were designed in the CBHI coding region and terminal subregion for PCR amplification to obtain Fragment 1. The primer sequences are as fo...

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Abstract

The invention discloses a Penicillium oxalicum host strain for enhancing expression of filamentous fungi protein, which is named Penicillium oxalicum M3QDelta15A and collected to China General Microbiological Culture Collection Center, wherein the collection number is CGMCC No.9092. The invention also discloses application of the strain in producing filamentous fungi protein. The experiment proves that the engineering bacterium disclosed by the invention has low secretion background in the aspect of expressing Penicillium oxalicum exoglucanase CBHI. Compared with the expression of host with Penicillium oxalicum strain Delta15A, the Penicillium oxalicum M3QDelta15A host has high protein expression level. When using cheap starch as the carbon source to produce the protein, the Penicillium oxalicum host strain has the advantages of low production cost and short period, and has favorable industrial development and application prospects.

Description

technical field [0001] The invention relates to an engineering bacterial strain with low extracellular secreted protein background on starch, in particular to a host bacterium for improving protein expression of filamentous fungi—Penicillium oxalicum (Penicillium oxalicum), which belongs to the field of genetic engineering. Background technique [0002] Protein expression system refers to the system composed of host, foreign gene, vector and auxiliary components. Through this system, the purpose of exogenous gene expression in the host can be achieved. The host expressing the protein is the organism expressing the protein, which can be bacteria, yeast, plant cells, animal cells, etc. Due to the different characteristics of various organisms, the types of proteins suitable for expression are also different. The most commonly used expression systems for heterologous expression of filamentous fungal proteins, especially glycoside hydrolases, are prokaryotic protein expression...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N1/15C12P21/00C12R1/80
Inventor 宋欣胡益波
Owner SHANDONG UNIV
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