Tackling destructive proteolysis of unconventionally secreted heterologous proteins in Ustilago maydis

Publication date: Available online 29 July 2018Source: Journal of BiotechnologyAuthor(s): Marius Terfrüchte, Sandra Wewetzer, Parveen Sarkari, Daniel Stollewerk, Mirita Franz-Wachtel, Boris Macek, Tino Schlepütz, Michael Feldbrügge, Jochen Büchs, Kerstin SchipperAbstractThe eukaryotic microorganism Ustilago maydis is currently being developed as an alternative protein expression platform. Protein fusion with an unconventionally secreted chitinase mediates export of heterologous proteins. The unique feature of this pathway is the circumvention of N-glycosylation. Different heterologous proteins could already be secreted via this novel mechanism in their active state. However, the system still suffers from low yields mainly attributed to the degradation of exported recombinant proteins by proteases. Here, we combined optimization steps on the level of cultivation conditions and strain engineering to further improve the system. Using the Respiration Activity Monitoring System we discovered that a pH drop during prolonged incubation results in loss of activity and degradation of the target protein. This problem can be reduced by buffering the cultivation medium. However, we still observed significant proteolysis even in buffered cultures. Hence, we revisited strain engineering to reduce the proteolytic activity. Secreted proteases were discovered using mass spectrometry. Then, genes for three identified proteases of a serine-carboxypeptidase family were deleted in an existing...

Source: Journal of Biotechnology - July 29, 2018 Category: Biotechnology Source Type: research