P74: Ashbya gossypii as a new fungal expression host for secretion of proteins

From an industrial point of view, protein production and secretion capabilities of the host systems currently in use are still insufficient. Different strategies can be utilized in this respect, and among them are: selection of the optimal host, improvement of the heterologous gene expression levels and reduction of the proteolytic activity in secretion vesicles. The filamentous hemiascomycete Ashbya gossypii has been proposed as a new player in biotech processes. A. gossypii can use a wide variety of carbon sources, grows to a very high cell density in simple media, it is considered as a GRAS organism and  shows low extracellular protease activities. Moreover only a few discrete bands of secreted proteins appear under normal cultivation conditions. In this work we have explored the potential of A. gossypii as a host for the expression and secretion of recombinant proteins. Using different A. gossypii promoters and signal sequences we were able to direct the secretion of three model proteins (E. coli phytase, human interleukin-1β and Candida rugosa lipase CRL1). Recombinant strains carrying the integrated expression cassette produced higher titers of secreted proteins than those based on yeast autonomously replicating plasmids. Although interleukin-1 β was scarcely produced and could be only detected by Western blot using monoclonal antibodies, both phytase and lipase were secreted as predominant catalytically active enzymes. In comparison with S. cerevisiae, A. gossypii showed much higher specific production of recombinant proteins providing the opportunity for additional improvements of this filamentous fungus as a host for the production of secreted proteins.