Abstract
Thermostable proteins show increased shelf life and performance at elevated temperatures and under harsh conditions, resulting in lower costs for various industrial and biotechnological applications. However, due to a limited understanding of the relationship between stability and function, protein stabilization remains primarily a trial-and-error approach. Therefore, building a combinatorial library of mutations predicted to improve stability, followed by experimental testing, represents a markedly improved methodology. However, the lack of high-throughput approaches to screen even a moderately sized library presents a major bottleneck in the field. Here, we use a thermophile, Parageobacillus thermoglucosidasius (Ptherm) to rapidly screen combinatorial libraries consisting of rationally designed thermostabilizing mutations (∼103–104) of a mesophilic fluorescent reporter, Y-FAST. On a Petri dish, microbial growth at an elevated temperature and exposure to fluorogen yielded several colonies of Ptherm that showed distinct fluorescence at 55 and 68 °C in our two sequentially generated libraries using Rosetta and ProteinMPNN, respectively. The Y-FAST variants isolated from fluorescent colonies were brighter than Y-FAST and showed higher resistance to thermal and chemical denaturation. AlphaFold-predicted structures and MD simulations revealed stability-enhancing salt bridges and hydrogen bond networks in the isolated FAST variants. The moderately thermostable FAST (tsFAST) and hyperstable FAST (hsFAST) were then demonstrated as translation reporters for protein expression and folding at elevated temperatures, such as 55 and 68 °C. Our approach of combinatorial library generation and high-throughput screening in a thermophilic chassis could, in principle, be extended to other proteins fused to these translation reporters. Furthermore, the hsFAST protein is small─half the size of the green fluorescent protein─and does not require oxygen for maturation, making it ideal for engineering extremophilic anaerobes for biosensing and bioconversion.
| Original language | English |
|---|---|
| Pages (from-to) | 4100-4115 |
| Number of pages | 16 |
| Journal | ACS Synthetic Biology |
| Volume | 14 |
| Issue number | 10 |
| DOIs | |
| State | Published - Oct 17 2025 |
Funding
This work was performed as part of the Bio-Optimized Technologies to keep Thermoplastics out of Landfills and the Environment (BOTTLE) Consortium, supported by DOE-EERE’s Bioenergy Technologies Office (BETO) and Advanced Materials and Manufacturing Technologies Office (AMMTO) under contract nos. NL0035994 and NL0037843, respectively. The work in part was also funded by the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office (BETO) for the Agile BioFoundry under contract NL0032182. Some part of the work was also conducted under Laboratory Directed Research and Development-funded project at Los Alamos National Laboratory under the project 20240306ER. The work was authored under Triad National Security, LLC (“Triad”) Contract No. 89233218CNA000001 with the U.S. Department of Energy. This research used computational resources provided by the Los Alamos National Laboratory Institutional Computing Program (under w21_proteng to R.K.J.), which is supported by the U.S. Department of Energy National Nuclear Security Administration under Contract No. 89233218CNA000001. This material is in part based upon work supported by the Center for Bioenergy Innovation (CBI), U.S. Department of Energy, Office of Science, Biological and Environmental Research Program under Award Number ERKP886. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. DOE under contract DE-AC05-00OR22725. This work is released for publication in accordance with LANL LA-UR-25-25037.
Keywords
- ProteinMPNN
- ROSETTA
- Y-FAST
- high-throughput screening
- protein engineering
- thermophile
- thermostability
- translational reporter