YTK Display-and-Secrete: Screening for Optimal Protein Secretion Elements in Saccharomyces cerevisiae

Area of Science:

• Biotechnology
• Synthetic Biology
• Molecular Biology

Background:

• Engineering yeast for efficient protein secretion is crucial for biotechnology.
• Current methods for optimizing protein secretion are often laborious and low-throughput.
• Identifying optimal promoter and signal peptide combinations is key for high expression and secretion.

Purpose of the Study:

• To develop a novel modular, high-throughput screening method for yeast strains designed for protein secretion.
• To enable rapid identification and optimization of genetic elements controlling protein secretion.
• To provide insights into optimal element choices for efficient protein secretion.

Main Methods:

• Integration of combinatorial DNA assembly, yeast surface display, flow cytometry, and nanopore DNA sequencing.
• Utilizing a yeast toolkit (YTK) for modular cloning and genome integration in *Saccharomyces cerevisiae*.
• Employing flow cytometry with fluorescently labeled antibodies for sorting high-secreting strains and pooled long-read sequencing for analyzing genetic element combinations.

Main Results:

• Demonstrated optimization of beta-lactamase and elastin-like polypeptide (ELP) secretion.
• Identified specific modular genetic element combinations that enhance protein surface display and subsequent secretion.
• Pooled sequencing revealed the effectiveness of numerous promoter and signal peptide combinations in a single experiment.

Conclusions:

• The developed system offers a powerful tool for high-throughput screening of yeast protein secretion.
• This method significantly accelerates the discovery of optimal genetic elements for enhanced protein production.
• The generated data can inform machine learning models for further optimization of protein secretion engineering.