As a eukaryote, U. maydis mainly exports proteins via the conventional secretion pathway involving the endoplasmic reticulum and the Golgi apparatus. However, an additional, unconventional secretion pathway has been identified recently for the chitinase Cts1. This route is of particular interest because it circumvents the endoplasmic reticulum and hence, N-glycosylation. Initial studies demonstrated that Cts1 can deal as a carrier to export heterologous proteins to the culture medium. Compared to conventional secretion this strategy allows the production of proteins lacking N-glycosylation in a eukaryotic system as well as the secretion of huge proteins. However, to date, yields of the system were limiting. Therefore, in UstiOpt the cultivation conditions were optimized for the enhanced production of recombinant proteins via unconventional secretion.
Initially, standard cultivations of a reporter strain for unconventional secretion were characterized in detail using multiple on- and off-line parameters. The analyses suggested that the pH during cultivation is critical to the yield of active and stable protein. Indeed, buffering of the medium led to a strong increase in reporter activity in the culture supernatant. In parallel, the influence of secreted proteases was evaluated, demonstrating that these contribute to protein degradation. Hence, the combination of buffered cultivation medium with a protease-deficient expression strain now strongly enhanced the yield of active full-length protein. Importantly, these novel insights provide the basis to produce biotechnological relevant proteins like biopharmaceuticals by unconventional secretion in competitive amounts.
Participating Core Groups
Dr. K. Schipper, Institute for Microbiology, Heinrich-Heine University Düsseldorf,
Building 26.12.01 Room 64
Dr. K. Schipper, Prof. Dr. M. Feldbrügge, Institute for Microbiology, HHU Düsseldorf
Dr. T. Schlepütz , Prof. Dr.-Ing. J. Büchs, Aachener Verfahrenstechnik, RWTH Aachen
01.03.2015 - 31.03.2016
UstiOpt is part of the NRW-Strategieprojekt BioSC and thus funded by the Ministry of Innovation, Science and Research of the German State of North Rhine-Westphalia.
Pompa, A, De Marchis, F, Pallotta, MT, Benitez-Alfonso, Y, Jones, A, Schipper, K, Moreau, K, Zarsky, V, Di Sansebastiano, GP and Bellucci, M (2017). Unconventional transport routes of soluble and membrane proteins and their role in developmental biology. Int J Mol Sci 18(4).
Sarkari, P, Feldbrügge, M and Schipper, K (2016). The corn smut fungus ustilago maydis as an alternative expression system for biopharmaceuticals. Gene expression systems in fungi: Advancements and applications. Schmoll, M. and Dattenböck, C. Cham, Springer International Publishing: 183-200.
Stock, J, Terfruchte, M and Schipper, K (2016). A reporter system to study unconventional secretion of proteins avoiding n-glycosylation in ustilago maydis. Methods Mol Biol 1459: 149-160.
Terfrüchte, M, Reindl, M, Jankowski, S, Sarkari, P, Feldbrügge, M and Schipper, K (2017). Applying unconventional secretion in ustilago maydis for the export of functional nanobodies. International Journal of Molecular Sciences 18(5): 937.