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

Microbial Cell Factories

Research

Franziska Wanka¹  Vera Meyer¹  Timothy C. Cairns¹  Arthur F. J. Ram²  Mark Arentshorst²  Thomas Jørgensen³ 

[1] Department Applied and Molecular Microbiology, Institute of Biotechnology, Berlin University of Technology, Gustav-Meyer-Allee 25, 13355, Berlin, Germany;Department Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands;Protein Expression, Novo Nordisk, Novo Nordisk Park, 2760, Måløv, Denmark;

关键词: Perfusion cultivation;    Zero growth rate;    Antifungal protein;    Hydrophobin;    Promoter;    Aspergillus niger;   

DOI  :  10.1186/s12934-016-0543-2

 received in 2016-03-04, accepted in 2016-08-08,  发布年份 2016

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY   
© The Author(s) 2016