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A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

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YESS 2.0, a Tunable Platform for Enzyme Evolution, Yields Highly Active TEV Protease Variants.

Carl A Denard¹, Chelsea Paresi¹, Rasha Yaghi¹

¹Department of Chemistry, University of Texas at Austin, Austin, Texas 78712, United States.

ACS Synthetic Biology

|January 6, 2021

Summary

We developed YESS 2.0 for engineering enzymes like proteases. This improved system enabled the creation of eTEV, the fastest Tobacco Etch Virus (TEV) protease variant yet, enhancing enzyme engineering capabilities.

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Area of Science:

Biochemistry
Molecular Biology
Enzyme Engineering

Background:

The original Yeast Endoplasmic Sequestration Screening (YESS) system facilitates enzyme characterization.
Engineering novel enzyme activities requires versatile screening platforms.

Purpose of the Study:

To introduce YESS 2.0, an advanced system for engineering and characterizing protein/peptide modifying enzymes.
To demonstrate YESS 2.0's utility by developing a highly efficient Tobacco Etch Virus (TEV) protease variant.

Main Methods:

YESS 2.0 incorporates modulated gene transcription and spatial sequestration of substrates and enzymes.
Protein engineering and structural modeling were used to enhance TEV protease activity.
Catalytic efficiency was assessed by measuring turnover rate (kcat) and substrate digestion.

Main Results:

YESS 2.0 offers a significantly higher operational and dynamic range than the original YESS.
A new TEV protease variant, eTEV, was engineered with 2.25-fold higher catalytic efficiency.
eTEV demonstrates rapid and specific digestion of fusion proteins at low enzyme-to-substrate ratios.
Structural modeling suggests enhanced catalytic efficiency is due to improved enzyme-substrate interactions.

Conclusions:

YESS 2.0 is a versatile platform for advanced enzyme engineering.
The eTEV variant represents a significant advancement in TEV protease technology.
Engineered proteases using YESS 2.0 have broad applications in biotechnology and research.