Quantification of product-specific gene expression in biopellets of Aspergillus niger with real-time PCR

The filamentous fungus Aspergillus niger is widely used as a host organism for homologous and heterologous protein production. A thorough characterization of product formation is needed to optimize the cultivation process. To evaluate the success of product formation during the cultivation, it is necessary to draw conclusions about the product yield on the genetic level. The method presented here includes the disruption of fungal cells, total RNA purification, reverse transcription of mRNA into cDNA and specific mRNA quantification by means of real-time PCR (polymerase chain reaction) using iCycler iQ and Light Upon eXtension (LUX™) primer technology. Specific mRNA quantification is based on the application of external standards, using total cellular RNA concentration as a reference. Quantitative real-time PCR has not been used for such purposes before. The method has been developed to monitor bioprocesses related to product formation. For the investigation presented, the protein glucoamylase has been used as a model product. The method allows the specific genetic activity of the cultivated pellets to be determined and can be modified for every other product. The results, which were generated during different fermentation experiments using the established method, very convincingly show the time at which glucoamylase was produced and corresponding mRNA was present in the fungal pellets.