Combinatorial Optimization of Promoters and Anchoring Proteins Drives a 10-Fold Enhancement in Yeast Surface Display of eGFP

Abstract

Yeast surface display (YSD) is a powerful tool for protein engineering, yet its broader application is often limited by suboptimal display efficiency. To address this, we systematically investigated the combinatorial effects of three key genetic determinants—promoter strength, anchoring protein identity, and fusion orientation (N- vs. C-terminal)—on the surface display level of enhanced green fluorescent protein (eGFP) in S. cerevisiae. A library of recombinant yeast strains was constructed, each harboring distinct combinations of these elements, and their display efficiencies were quantitatively assessed via fluorescence spectroscopy-based analysis. Our results demonstrate that the synergistic optimization of all three parameters is essential for maximizing YSD performance. Among all constructs tested, the novel plasmid system pYGAL1-Sed1p-eGFP-C—which combines the strong inducible GAL1promoter, the Sed1p cell wall anchor, and a C-terminal eGFP fusion—exhibited the highest display efficiency. This optimal configuration achieved a mean fluorescence intensity over 10.3-fold higher than the baseline system (pYSED1-Aga1p-eGFP-C) and 9.5-fold higher than the alternative N-terminal fusion design (pYSED1-eGFP-Aga1p-N). These findings establish a clear design principle for engineering high-efficiency YSD platforms. The pYGAL1-Sed1p-eGFP-C system not only provides a robust and highly efficient chassis for displaying eGFP but also holds significant promise as a versatile scaffold for the surface presentation of diverse heterologous proteins in yeast, thereby expanding the utility of YSD in biotechnology and synthetic biology.