ABSTRACT

Quantification of protein secretory expression at the single-cell level with low-cost, high-throughput screening approaches poses a great challenge. Here, we established a single-cell screening platform based on the HiBiT/ LgBiT complementation system in Pichia pastoris. In this platform, surface fluorescence serves as a proxy for secretion capacity, enables sensitive detection of surface-displayed target proteins, and is fully compatible with flow cytometry for high-throughput screening. By constructing an N-terminal random mutation library of recombinant human hyaluronidase PH20 (rHuPH20), multiple mutants with significantly enhanced secretion were obtained, with the highest enzyme activity approximately doubled compared to the wild-type. Sequence analysis suggested that these improvements were associated with alterations in N-terminal sequence features linked to translation initiation and early folding dynamics. In parallel, QTY-based rational design was applied to optimize the C-terminal membrane-associated region, reducing local hydrophobicity and facilitating protein export. rHuPH20 variants incorporating both N-terminal mutations and C-terminal QTY modification exhibited enhanced surface-associated signals and extracellular enzymatic activity relative to the wild-type. The bestperforming variant, (4-4)rHuPH20QTY, achieved an enzymatic activity of 0.098 U/mL, which represents a 1.51-fold increase compared to the wild-type (0.065 U/mL). Overall, this study presents an effective single-cell screening strategy based on a surface-display proxy for identifying secretion-enhancing mutants and provides insights into rational approaches for improving the secretion of challenging recombinant proteins in yeast.