Highly active promoters and native secretion signals for protein production during extremely low growth rates in Aspergillus niger

Abstract

BackgroundThe filamentous ascomycete Aspergillus niger is used in many industrial processes for the production of enzymes and organic acids by batch and fed-batch cultivation. An alternative technique is continuous cultivation, which promises improved yield and optimized pipeline efficiency.ResultsIn this work, we have used perfusion (retentostat) cultivation to validate two promoters that are suitable for A. niger continuous cultivation of industrially relevant products. Firstly, promoters of genes encoding either an antifungal protein (Panafp) or putative hydrophobin (PhfbD) were confirmed as active throughout retentostat culture by assessing mRNA and protein levels using a luciferase (mluc) reporter system. This demonstrated the anafp promoter mediates a high but temporally variable expression profile, whereas the hfbD promoter mediates a semi-constant, moderate-to-high protein expression during retentostat culture. In order to assess whether these promoters were suitable to produce heterologous proteins during retentostat cultivation, the secreted antifungal protein (AFP) from Aspergillus giganteus, which has many potential biotechnological applications, was expressed in A. niger during retentostat cultivation. Additionally, this assay was used to concomitantly validate that native secretion signals encoded in anafp and hfbD genes can be harnessed for secretion of heterologous proteins. Afp mRNA and protein abundance were comparable to luciferase measurements throughout retentostat cultivation, validating the use of Panafp and PhfbD for perfusion cultivation. Finally, a gene encoding the highly commercially relevant thermal hysteresis protein (THP) was expressed in this system, which did not yield detectable protein.ConclusionBoth hfbD and anafp promoters are suitable for production of useful products in A. niger during perfusion cultivation. These findings provide a platform for further optimisations for high production of heterologous proteins with industrial relevance.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0543-2) contains supplementary material, which is available to authorized users.

A Transcriptome Meta-Analysis Proposes Novel Biological Roles for the Antifungal Protein AnAFP in Aspergillus niger

“…When both strains were cultivated in microtiter plates, a peak in luciferase activity between 40–50 h of cultivation was observed for both strains, whereby strain NP6.4 showed about 6-fold higher expression levels compared to strain PK2.9, thus clearly demonstrating that the anafp promoter is under control of CreA (Fig 3A). Note that the observed peak-like temporal expression pattern is in good agreement with the anafp expression profile observed for retentostat cultivations (see above and [35]). We also generated reporter strain PK1.22, in which expression of the fluorescent protein eYFP was put under control of the anafp promoter.…”

Antifungal Peptides of the AFP Family Revisited: Are These Cannibal Toxins?

“…This hypothesis is supported by publications from others who demonstrated that asexual sporulation of A. nidulans is accompanied by autolytic and apoptotic processes [ 53 , 54 , 55 , 56 ]. It is also in agreement with the observation that there is a considerable delay between induction of anafp gene expression and detection of AnAFP in the culture supernatant of A. niger [ 44 , 57 ]. Some AFPs (e.g., PAFB from P. chrysogenum and AFPB from P. digitatum ) even remained undetectable in the medium although their encoding genes were transcribed at high levels [ 13 , 37 ].…”

Effect of CBM1 and linker region on enzymatic properties of a novel thermostable dimeric GH10 xylanase (Xyn10A) from filamentous fungus Aspergillus fumigatus Z5

“…The protein of Xyn10A had several similar homologs (> 70%) when blasted against the NCBI database ( http://www.ncbi.nlm.nih.gov ), however, it was firstly reported in this study as a high thermostable GH10 xylanase with an optimal temperature of 90 °C in Aspergillus species. It will be suitable for adding this thermostable GH10 xylanase into enzyme cocktails by expressing it in an efficient protein expression system of A. niger (Wanka et al 2016 ) or other industrial strains like T. reesei . If being considered, the potential of this enzyme still could be increased, such as enhancing the stability at high temperatures (> 80 °C), or improving the xylanase activity at low temperatures to make this enzyme work efficiently in a wide range of temperatures.…”