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

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biotechnology

Background:

• Efficient secretion of proteins and enzymes is vital for synthetic biology applications, enabling continuous production and purification without cell lysis.
• While N-terminal signal peptides (SPs) direct eukaryotic protein secretion, their sequence impact on efficiency is poorly understood, and current systems use limited SPs.
• Optimizing genetic constructs, including promoters and terminators, is critical for secretion efficiency but remains a bottleneck due to inefficient screening methods.

Purpose of the Study:

• To develop a high-throughput screening (HTS) method for optimizing protein secretion efficiency.
• To quantify the impact of signal peptide (SP) sequence variation on protein secretion.
• To identify optimal combinations of genetic elements for enhanced heterologous protein expression.

Main Methods:

• Adaptation of a yeast G-protein-coupled receptor (GPCR) biosensor to measure co-secreted peptide tags.
• Quantification of protein secretion efficiency via a fluorescent reporter linked to GPCR activation.
• Assembly of over 6000 genetic construct combinations using one-pot Combinatorial Golden Gate cloning for HTS.

Main Results:

• Demonstrated the biosensor's capability for rapid identification of optimal secretion element combinations.
• Successfully quantified protein secretion levels using the developed HTS system.
• Highlighted the significant impact of SP sequence optimization on secretion efficiency.

Conclusions:

• The developed yeast biosensor provides an efficient HTS platform for optimizing protein secretion.
• Signal peptide optimization is a critical initial step in designing effective heterologous protein expression strategies.
• This approach significantly enhances the design and efficiency of protein secretion systems in synthetic biology.