(48b) Protein Endocytosis and Degradation: Impact On Secreted Protein Titers in Yeast

Recombinant protein production is an important activity in the production of drugs, fuels, and chemicals.  Secreting protein into minimal media simplifies downstream purification, therefore reducing cost of manufacture.  To date, considerable effort has been placed on increasing protein titers through improving recombinant protein synthesis and secretion.  In the present work, we unexpectedly identified protein uptake and degradation as a significant process that decreases protein titers in Saccharomyces cerevisiae.  Experiments showing (1) competitive endocytosis of insulin, bovine serum albumin (BSA), and yeast extract protein and (2) growth on BSA as a sole carbon source was used establish endocytosis as the mechanism of protein loss.   Transcriptomics showed decreased amino acid synthesis, increased amino acid degradation/utilization, and metabolomics showed increased concentrations of amino acids, indicating large scale uptake of protein for carbon and energy.  We identify key mechanisms that can be further engineered to reduce the re-uptake of recombinant proteins.  This work has revealed an unanticipated mechanism for product loss that impact many industrial and pharmaceutical bioprocesses.