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Aspergillus niger strain for protein producing and application thereof

A technology of Aspergillus niger and Aspergillus niger, applied in the field of Aspergillus niger strain and its application in protein production

Active Publication Date: 2017-11-24
王枫枫
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, if it is possible to make Aspergillus niger achieve whole mycelium secretion to significantly improve the production efficiency of protein or enzyme preparations, there is no relevant report yet.

Method used

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  • Aspergillus niger strain for protein producing and application thereof
  • Aspergillus niger strain for protein producing and application thereof
  • Aspergillus niger strain for protein producing and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Construction and effect study of genetically engineered strain of Aspergillus niger with fluG gene knocked out

[0040] 1. The upstream and downstream sequences of fluG are amplified with A.niger N402 genomic DNA template, and the NotI and XbaI / XhoI restriction enzyme sites are introduced to the 5'and 3'ends of the upstream sequence through primers, while XhoI and KpnI was introduced into the 5'and 3'ends of the downstream sequence; the amplified primers were blunt-ended to the pJET 1.2 (Fermentas, Thermoscientific, Waltham, USA) vector; the resulting upstream and downstream fragment vectors were subjected to NotI / XhoI and XhoI / KpnI are digested, and the resulting fragments are recovered for later use; then use the vector pBluescriptIISK(+) as the backbone to link the three fragments; insert PAN7-1XhoI / XbaI enzyme slices between the upstream and downstream sequences of fluG, and this fragment contains hygromycine resistance Express gene; finally complete the con...

Embodiment 2

[0042] Example 2 Aspergillus niger mutant strain FY17 (ΔfluGamyR + ) Build

[0043] The amyR gene was overexpressed on the basis of the Aspergillus niger ΔfluG mutant prepared in Example 1. The specific implementation is as follows. A 4.3kb DNA fragment (SEQ ID NO. 2) containing the amyR gene was introduced into the pGEM11 vector and combined with pIM2101 (the The vector contains the argB gene) for co-transformation. The mutants obtained after transformation and screening are subjected to a series of further screening work on the starch substrate. The obtained strains are subjected to molecular testing, screening and confirmation of the inserted copy number, and finally the FY17 strain is obtained. The FY17 engineered strain of the present invention is based on the fluG mutant strain (ΔfluG), after adding 8-12 copies of amyR gene, it exhibits the ability of the whole hyphae hypersecretion of glucoamylase.

[0044] After introducing multiple copies of the glucoseamylase regulatory f...

Embodiment 3

[0048] Example 3 Aspergillus niger mutant strain (ΔfluGamyR + ΔpepA) construction

[0049] 1. Construction method

[0050] The upstream and downstream sequences of pepA were amplified using A.niger N402 genomic DNA template, and the NotI and XbaI / XhoI restriction enzyme sites were introduced through primers to the 5'and 3'ends of the upstream sequence, while XhoI and salI were Was introduced into the 5'and 3'ends of the downstream test sequence; the amplified primers were blunt-ended to pJET 1.2 (Fermentas, Thermoscientific, Waltham, USA) vector; the resulting upstream and downstream fragment vectors were subjected to NotI / XhoI and After digestion with XhoI / salI, the fragments obtained are recovered for later use. Next, use the vector pBluescriptIISK(+) as the backbone to link the three fragments; insert the pXDRFP4 (Yang et al. 2004) XhoI / XbaI enzyme slices between the upstream and downstream sequences of pepA. Contains pryG resistance expression gene; finally complete the constr...

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Abstract

The invention provides an aspergillus niger strain for protein producing and application thereof, which belong to the field of gene engineering. After a fluG gene is knocked out of the aspergillus niger strain, an obtained mutant strain has a protein high-yielding property; further, a saccharifying enzyme regulating factor is introduced into a mutant strain genome, and an obtained mutant strain FY17(delta fluGamyR<+>) can promote secretion of homologous proteins and heterologous proteins; after a pepA gene is knocked out of the FY17 mutant strain, an obtained mutant strain can also be beneficial for remarkably improving secretory volume of the homologous proteins and the heterologous proteins; the invention also discovers that the FY17 SP(delta fluGdeltapepAamyR<+>AnhapC<+>) can be used as an expression vector for producing a plurality of heterologous proteins. The mutant strains adopting the aspergillus niger strain without the fluG gene as an original strain have favorable capabilities on producing the homologous proteins and the heterologous proteins, so that the aspergillus niger strain has a favorable market application prospect.

Description

Technical field [0001] The invention belongs to the technical field of microbial genetic engineering modification, and specifically relates to an Aspergillus niger strain after genetic engineering modification and its application in protein production. Background technique [0002] The filamentous fungus Aspergillus niger, as a host for transformation, has the following advantages over other microorganisms such as bacteria and yeast: Aspergillus niger has a strong ability to secrete extracellular proteins, while the recombinant protein expressed by bacteria is often contained in inactive and insoluble When bacteria express heterologous eukaryotic genes, their translation and transcription are different from those of eukaryotic organisms, the transferred genes may not be expressed normally, which does not exist in filamentous fungi, and filamentous fungi can The expressed protein undergoes correct post-translational processing, including peptide-linked cleavage and glycosylation. ...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/80C12N15/67C12R1/685
CPCC07K14/38C12N9/62C12N15/67C12N15/80
Inventor 王枫枫王一丁
Owner 王枫枫
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