Thermophilic Chassis-Enabled High-Throughput Selection of a Thermostable Fluorogenic Reporter

Area of Science:

• Protein Engineering
• Biotechnology
• Structural Biology

Background:

• Thermostable proteins offer extended shelf life and improved performance in industrial settings, reducing costs.
• Current protein stabilization methods rely heavily on trial-and-error due to limited understanding of stability-function relationships.
• High-throughput screening is a bottleneck for evaluating large combinatorial protein libraries.

Purpose of the Study:

• To develop an improved methodology for protein stabilization by creating and screening combinatorial libraries of mutations.
• To establish a high-throughput screening approach in a thermophilic host for rapid identification of thermostable proteins.
• To engineer a fluorescent reporter protein with enhanced stability and functionality at elevated temperatures.

Main Methods:

• Utilized a thermophile, *Parageobacillus thermoglucosidasius* (*Ptherm*), as a chassis for high-throughput screening.
• Generated and screened combinatorial libraries (∼10^3-10^4) of rationally designed thermostabilizing mutations for the Y-FAST reporter.
• Employed microbial growth at elevated temperatures (55 and 68 °C) and fluorogen exposure for screening.
• Validated Y-FAST variants using AlphaFold structure prediction and molecular dynamics (MD) simulations.

Main Results:

• Successfully isolated Y-FAST variants (tsFAST and hsFAST) exhibiting enhanced fluorescence and stability at 55 and 68 °C.
• Isolated variants demonstrated superior resistance to thermal and chemical denaturation compared to the original Y-FAST.
• Structural analysis revealed stability-enhancing salt bridges and hydrogen bond networks in the engineered variants.
• Demonstrated tsFAST and hsFAST as effective translation reporters for protein expression and folding at high temperatures.

Conclusions:

• The combinatorial library generation and high-throughput screening in a thermophilic chassis is an effective strategy for protein engineering.
• This approach can be extended to other proteins and thermophilic hosts for various biotechnological applications.
• The small, oxygen-independent hsFAST protein is suitable for engineering extremophilic anaerobes for biosensing and bioconversion.