High-throughput optimisation of protein secretion in yeast via an engineered biosensor

Secretion of high-value proteins and enzymes is fundamental to the synthetic biology economy, allowing continuous fermentation during production and protein purification without cell lysis. Most eukaryotic protein secretion is encoded by an N-terminal signal peptide (SP); however, the strong impact of SP sequence variation on the secretion efficiency of a given protein is not well defined. Despite high natural SP sequence diversity, most recombinant protein secretion systems use only a few well-characterised SPs. Additionally, the selection of promoters and terminators can significantly affect secretion efficiency, yet screening numerous genetic constructs for optimal sequences remains inefficient. Here, we adapted a yeast G-protein-coupled receptor (GPCR) biosensor, to measure the concentration of a peptide tag that is co-secreted with any protein of interest (POI). Thus, protein secretion efficiency can be quantified via induction of a fluorescent reporter that is upregulated downstream of receptor activation. This enabled high-throughput screening of over 6000 combinations of promoters, SPs, and terminators, assembled using one-pot Combinatorial Golden Gate cloning. We demonstrate this biosensor can quickly identify best combinations for secretion and quantify secretion levels. Our results highlight the importance of SP optimisation as an initial step in designing heterologous protein expression strategies, demonstrating the value of high-throughput screening (HTS) approaches for maximising secretion efficiency.