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Promoter for filamentous fungal protein secretion pressure feedback regulation element and feedback inhibition resistance, plasmid, preparation method and transformed cell

A technology of filamentous fungi and regulatory elements, applied in the field of genetic engineering

Active Publication Date: 2013-09-04
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there has been no report on the cis-acting regulatory elements of the RESS mechanism of the amyB gene promoter

Method used

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  • Promoter for filamentous fungal protein secretion pressure feedback regulation element and feedback inhibition resistance, plasmid, preparation method and transformed cell
  • Promoter for filamentous fungal protein secretion pressure feedback regulation element and feedback inhibition resistance, plasmid, preparation method and transformed cell
  • Promoter for filamentous fungal protein secretion pressure feedback regulation element and feedback inhibition resistance, plasmid, preparation method and transformed cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] 1. Extraction of pTA1110 and pPTRI plasmid DNA

[0040] The plasmid pTA1110 containing the full-length amyB gene promoter, the reporter gene rml and the agdA gene terminator and the plasmid pPTRI (purchased from Takara Company) with PT (pyrithiamine) resistance can be extracted using a commercially available plasmid extraction kit.

[0041] 2. Construction of fragmented promoters and fusion-deleted promoters

[0042] 2.1 Construction of Fragmented Promoters

[0043] According to the reported Aspergillus oryzae amyB gene promoter sequence, primers were designed to transform the amyB gene promoter. Fragmented promoter primers were designed to sequentially truncate the amyB gene promoter into 14 different gene expression units from the 5' end: P amyB941 (Promoter region -941bp~-1bp, SEQ ID NO.9 and SEQ ID NO.7); P amyB871 (Promoter region -871bp~-1bp, SEQ ID NO.10 and SEQ ID NO.7); P amyB766 (Promoter region -766bp~-1bp, SEQ and SEQ ID NO.7); P amyB674 (Promoter regio...

Embodiment 2

[0070] 1. Fermentation of Aspergillus oryzae transformants

[0071] Will P △F378-291 promoter and P amyB1110 The Aspergillus oryzae transformants AO3821 and AO1110 of the expression unit constructed by the promoter and rml were inoculated into 50mL (500mL baffle shake flask) fermentation medium (0.3% peptone, 0.2% KCl, 0.05% MgSO 4 .7H 2 O, 0.001% FeSO 4 .7H 2 O, 0.1% K 2 PHO 4 , 2% maltose, pH5.5), the final concentration of spores reached 10 6 pieces / ml. Ferment at 30°C and 200r / min.

[0072] 2. Determination method of enzyme activity

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Abstract

The invention discloses a promoter for a filamentous fungal protein secretion pressure feedback regulation element and feedback inhibition resistance, a plasmid, a preparation method and a transformed cell. The regulation element is a base sequence of the area from (-378bp)-(-291bp) of a filamentous fungal promoter. The regulation element is related to the protein secretion pressure feedback. The promoter against feedback inhibition is a base sequence obtained by at least losing or replacing the base of the area from (-378bp)-(-291bp) on the filamentous fungal promoter, and has promoter activity. The promoter disclosed by the invention can be efficiently expressed in filamentous fungi and particularly in the protein of aspergillus oryzae.

Description

technical field [0001] The invention belongs to the field of genetic engineering. Specifically, the present invention relates to a cis-element associated with the feedback regulation of protein secretion pressure in filamentous fungi, and a cis-element that is released in filamentous fungi such as Aspergillus oryzae due to the absence of a protein secretion pressure feedback cis-regulatory element. Promoter for highly expressed proteins that are feedback-repressed by secretion pressure. Background technique [0002] Filamentous fungi have a strong ability to secrete proteins, and can correctly process and modify proteins after translation. Their glycosylation pathways are close to those of mammals. In particular, their Aspergillus species have long been used in traditional food and beverage industries, and are recognized as safe Production strains are ideal host bacteria for industrial production of recombinant proteins (Nevalainen KM et al., 2005). However, compared with ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63C12N15/66C12N1/14C12R1/69C12R1/685C12R1/665C12R1/66
Inventor 潘力周斌王超
Owner SOUTH CHINA UNIV OF TECH
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