Yeast Surface Display For Enzyme Engineering Service

Yeast Surface Display (YSD) is a robust and quantitative platform for the directed evolution and engineering of enzymes, particularly those that are large, complex, or require post-translational modifications. In YSD, the enzyme variant is fused to an anchor protein (typically Aga2p) and displayed on the cell wall of the yeast Saccharomyces cerevisiae. This system allows for the display of large proteins, offers eukaryotic folding machinery (including disulfide bond formation), and is compatible with fluorescent-activated cell sorting (FACS) for highly quantitative and rapid screening.

CD Biosynsis offers comprehensive CRO services in Yeast Surface Display for enzyme engineering, providing a highly scalable method to screen vast enzyme libraries (up to 10^8 variants) with exceptional precision. Our platform is ideal for tailoring enzyme properties such as substrate affinity, catalytic efficiency, and stability. We utilize multi-parameter FACS sorting with fluorescently-labeled substrates or inhibitors to select for subtle functional differences between variants. YSD's compatibility with eukaryotic expression makes it the superior choice for engineering complex enzymes and optimizing them directly in a host environment relevant to industrial biomanufacturing.

Highlights

YSD provides a high-fidelity, quantitative method for evolving complex enzymes and proteins.

• Quantitative Screening via FACS: Permits the precise quantification and sorting of cells based on enzyme activity or binding affinity using fluorescent labeling, achieving high resolution.
• Eukaryotic Protein Folding: Supports the correct folding, disulfide bond formation, and post-translational modification (PTMs) of complex enzymes, unlike prokaryotic systems.
• High Library Capacity: Capable of screening large libraries (up to 10^8) in a short time frame, effectively exploring the sequence space for superior mutants.
• Affinity and Activity Screening: Allows simultaneous measurement of both the enzyme display level and its functional activity, enabling selection based on specific activity (activity/display level).

Applications

YSD is a powerful tool for engineering complex or therapeutically relevant enzymes:

Engineering Complex Enzymes

Optimizing large enzymes, multi-domain proteins, or those requiring complex PTMs, which fail to fold correctly in E. coli display systems.

Affinity Maturation

Generating enzyme variants with dramatically improved substrate binding affinity (lower Km) or inhibitor binding (lower Ki) through iterative sorting rounds.

Stereoselectivity Enhancement

Using fluorescently-labeled chiral substrates to quantitatively sort for mutants that display the highest enantiomeric ratio in their catalytic output.

Library Screening for New Functions

Rapidly screening large, randomly generated libraries to discover enzymes with novel catalytic activities toward non-native or toxic substrates.

Platform

Our YSD platform is built around high-precision FACS sorting and robust library construction.

Fluorescence-Activated Cell Sorting (FACS)

Utilization of high-speed FACS for quantitative analysis and sorting of single yeast cells based on two or more fluorescence signals (e.g., binding and display level).

Synthetic Library Generation

Construction of high-diversity enzyme libraries (10^6 - 10^8 variants) using techniques like error-prone PCR, DNA shuffling, or combinatorial saturation mutagenesis.

Multi-Parameter Sorting

Simultaneous use of anti-Aga2p antibodies (to measure display level) and fluorescent ligands/substrates (to measure function), ensuring selection of the most active variant, not just the best displayed.

Kinetic Selection

Implementing off-rate screening protocols (using competitive elution or long washing steps) to select variants with extremely slow dissociation rates (high affinity/stability).

High-Content Validation

Post-sorting functional validation of isolated clones using purified protein kinetics (Km, kcat) and biophysical stability analysis (Tm).

Workflow

Our Yeast Surface Display workflow employs a tightly controlled, iterative loop for directed evolution:

• Library Construction: Generate the enzyme mutant library (e.g., via PCR) and clone it into the yeast display vector, ensuring fusion with the Aga2p anchor protein.
• Yeast Transformation and Display Induction: Transform the yeast (S. cerevisiae) and induce expression of the enzyme variants on the cell surface.
• Fluorescent Labeling: Label the yeast cells using two fluorescent probes: one targeting the Aga2p display tag (to quantify total display) and one targeting the functional property (e.g., fluorescent substrate binding/turnover).
• FACS Sorting (Panning): Run the labeled cells through the FACS machine. Sort the top fraction (e.g., 0.1%) showing high functional signal per unit of display signal (high specific activity).
• Mutant Recovery and Amplification: Recover the sorted yeast cells and amplify the plasmid DNA encoding the successful variants through growth/culturing.
• Iterative Rounds and Analysis: Repeat the labeling and sorting steps (typically 3-4 rounds) to maximize enrichment. Sequence the final enriched pool and characterize the purified protein hits.

CD Biosynsis delivers functionally superior enzyme variants with comprehensive characterization data. Every project includes:

• Design Report: Details of the library size, FACS gating strategy, and enrichment statistics across all rounds.
• Engineered Variant Sequences: DNA and protein sequences of the best-performing enzyme mutants confirmed by Sanger or NGS sequencing.
• Functional Kinetics: Purified protein kinetic data (Km, kcat, kcat/Km) comparing the top mutant to the wild-type enzyme.
• Expression Vectors: Delivery of the plasmid containing the optimized enzyme for straightforward purification and scale-up.