Yeast Synthetic Minimal Biosensors for Evaluating Protein Production

The unfolded protein response (UPR) is a highly conserved cellular response in eukaryotic cells to counteract endoplasmic reticulum (ER) stress, typically triggered by unfolded protein accumulation. In addition to its relevance to human diseases like cancer cell development, the induction of the UPR has a significant impact on recombinant protein production yields in microbial cell factories, including the industrial workhorse Saccharomyces cerevisiae. Being able to accurately detect and measure this ER stress response in single cells, enables the rapid optimisation of protein production conditions and high-throughput strain selection strategies. Current methodologies to monitor the UPR in S. cerevisiae are often temporally and spatially removed from the cultivation stage, or lack updated systematic evaluation. To this end we constructed and systematically evaluated a series of high-throughput UPR sensors by different designs, incorporating either yeast native UPR promoters or novel synthetic minimal UPR promoters. The native promoters of DER1 and ERO1 were identified to have suitable UPR biosensor properties and served as an expression level guide for orthogonal sensor benchmarking. Our best synthetic minimal sensor, SM1, was only 98 bp in length, had minimal homology to other native yeast sequences and displayed superior sensor characteristics. Using this synthetic minimal UPR sensor, we demonstrate its ability to accurately discriminate between cells expressing different heterologous proteins and at varying production levels. Our sensor is thus a novel high-throughput tool for determining expression/engineering strategies for optimal heterologous protein production.