A kind of aspergillus niger bacterial strain that can be used for protein production and application thereof

A technology of Aspergillus niger strains, Aspergillus niger, applied in the directions of microorganism-based methods, introduction of foreign genetic material, fungi, etc. using vectors

Active Publication Date: 2021-03-23
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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  • A kind of aspergillus niger bacterial strain that can be used for protein production and application thereof
  • A kind of aspergillus niger bacterial strain that can be used for protein production and application thereof
  • A kind of aspergillus niger bacterial strain that can be used for protein production and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Construction and effect research of the Aspergillus niger genetically engineered strain that knocked out the fluG gene

[0040] 1. FluG upstream and downstream sequences are amplified with the A.niger N402 genomic DNA template, and primers are used to introduce NotI and XbaI / XhoI restriction endonuclease sites to the 5' and 3' ends of the upstream sequence, while XhoI and KpnI was then introduced into the 5' and 3' ends of the downstream sequence; the amplified primers were blunt-ended connected to the pJET 1.2 (Fermentas, Thermoscientific, Waltham, USA) vector; the resulting upstream and downstream fragment vectors were subjected to NotI / Digested with XhoI and XhoI / KpnI, the resulting fragments were recovered for later use; next, the vector pBluescriptIISK(+) was used as the backbone to link the three fragments; the upstream and downstream sequences of fluG were inserted into the PAN7-1XhoI / XbaI enzyme fragment, which contained hygromycine resistance Express...

Embodiment 2

[0042] Embodiment 2 Aspergillus niger mutant strain FY17 (ΔfluGamyR + ) construction

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

[0044] After introducing multiple copies of the glucoseamyla...

Embodiment 3

[0048] Embodiment 3 Aspergillus niger mutant strain (ΔfluGamyR + Construction of ΔpepA)

[0049] 1. Construction method

[0050] pepA upstream and downstream sequences were amplified with the A.niger N402 genomic DNA template, and primers introduced NotI and XbaI / XhoI restriction endonuclease sites to the 5' and 3' ends of the upstream sequence, while XhoI and salI were Introduced into the 5' and 3' ends of the downstream sequence; the amplified primers were blunt-ended connected to the pJET 1.2 (Fermentas, Thermoscientific, Waltham, USA) vector; the resulting upstream and downstream fragment vectors were carried out for NotI / XhoI and Digest with XhoI / salI, and recover the resulting fragments for later use; then use the vector pBluescriptIISK(+) as the backbone to link the three fragments; insert pXDRFP4 (Yang et al. Contains the pryG resistance expression gene; finally completes the construction of the pepA gene knockout plasmid pepAKO. FY17 was used as the host strain for...

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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 transformation, and in particular relates to a genetically engineered Aspergillus niger strain and its application in protein production. Background technique [0002] The filamentous fungus Aspergillus niger, as the host for transformation, has the following advantages over other microorganisms such as bacteria and yeasts: Aspergillus niger has a strong ability to secrete proteins outside the cell, while recombinant proteins expressed by bacteria often contain inactive insoluble When bacteria express heterologous eukaryotic genes, because their translation and transcription are different from those of eukaryotes, the transferred genes may not be expressed normally, but this situation does not exist in filamentous fungi. Filamentous fungi can The expressed protein undergoes correct post-translational processing, including post-processing such as peptide cleavage and glycosyla...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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