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. 2016 Apr 28;15:68.
doi: 10.1186/s12934-016-0463-1.

The amyR-deletion Strain of Aspergillus Niger CICC2462 Is a Suitable Host Strain to Express Secreted Protein With a Low Background

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Free PMC article

The amyR-deletion Strain of Aspergillus Niger CICC2462 Is a Suitable Host Strain to Express Secreted Protein With a Low Background

Hui Zhang et al. Microb Cell Fact. .
Free PMC article

Abstract

Background: The filamentous fungus Aspergillus niger is widely exploited as an important expression host for industrial production. The glucoamylase high-producing strain A. niger CICC2462 has been used as a host strain for the establishment of a secretion expression system. It expresses recombinant xylanase, mannase and asparaginase at a high level, but some high secretory background proteins in these recombinant strains still remain, such as alpha-amylase and alpha-glucosidase; lead to a low-purity of fermentation products. The aim was to construct an A. niger host strain with a low background of protein secretion.

Results: The transcription factor amyR was deleted in A. niger CICC2462, and the results from enzyme activity assays and SDS-PAGE analysis showed that the glucoamylase and amylase activities of the ∆amyR strains were significantly lower than those of the wild-type strain. High-throughput RNA-sequencing and shotgun LC-MS/MS proteomic technology analysis demonstrated that the expression of amylolytic enzymes was decreased at both the transcriptional and translational levels in the ∆amyR strain. Interestingly, the ∆amyR strain growth rate better than the wild-type strain.

Conclusions: Our findings clearly indicated that the ∆amyR strain of A. niger CICC2462 can be used as a host strain with a low background of protein secretion.

Keywords: Aspergillus niger; Proteomics; Regulation; Transcriptome; amyR.

Figures

Fig. 1
Fig. 1
a Activities of amylolytic enzymes from A. niger CICC2462 and ∆amyR (amyR3, amyR6 and amyR12) strains grown on fermentation medium. b Separation of identified amylolytic enzyme proteins from the fermentation supernatant of A. niger CICC2462 and ∆amyR (amyR3, amyR6 and amyR12) strains by SDS-PAGE. 1, 2 and 3 represent glucoamylase, acid-stable alpha-amylase and alpha-amylase, respectively. An volume of 10 μL of each sample was separated on a 12 % resolving gel. c The protein concentration of A. niger CICC2462 and amyR12 strains
Fig. 2
Fig. 2
Quantitative real-time PCR of some representative transcripts that were differentially expressed in A. niger CICC2462 and ∆amyR (amyR3, amyR6 and amyR12) strains grown on fermentation medium. The selected genes were: glucoamylase gene glaA, amylase gene amyA and transcriptional activator amyR
Fig. 3
Fig. 3
Activities of protease activities from A. niger CICC2462 and ∆amyR strains grown on fermentation medium
Fig. 4
Fig. 4
a Growth of A. niger CICC2462 and ∆amyR strains on PDA medium. b Growth of A. niger CICC2462 and ∆amyR strains on PDA medium after 8 days. c Biomass weights of A. niger CICC2462 and ∆amyR grown in fermentation medium
Fig. 5
Fig. 5
Quantitative real-time PCR of creA gene that was differentially expressed in A. niger CICC2462 and amyR12 strains grown on fermentation medium

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