# Screening of molecular elements and improvement of heat resistance in a thermophilic bacterium

## TL;DR

Researchers engineered a heat-resistant bacterium by combining genetic modifications and genome reduction, enabling it to survive at high temperatures.

## Contribution

The study introduces a novel strain of Geobacillus stearothermophilus with enhanced thermotolerance and a new electroporation method for the genus.

## Key findings

- Synergistic overexpression of heat-associated genes increased survival to 85 °C.
- Genome reduction and adaptive evolution improved stability at 80 °C.
- Electroporation efficiency reached 10⁴ CFU/µg DNA, enabling high-temperature protein expression.

## Abstract

•Engineered G. stearothermophilus SL-1-H1 survives at 85 °C via gene overexpression.•Developed first efficient electroporation for Geobacillus (10⁴ CFU/µg DNA).•Synergistic gene overexpression (e.g., GroES-GrpE) boosted thermotolerance.•ALE & genome reduction created base mutant (SL-1–80) stable at 80 °C.•Genomic changes promoted motility, metabolism & stress response under heat.

Engineered G. stearothermophilus SL-1-H1 survives at 85 °C via gene overexpression.

Developed first efficient electroporation for Geobacillus (10⁴ CFU/µg DNA).

Synergistic gene overexpression (e.g., GroES-GrpE) boosted thermotolerance.

ALE & genome reduction created base mutant (SL-1–80) stable at 80 °C.

Genomic changes promoted motility, metabolism & stress response under heat.

Engineering microorganisms to withstand extreme temperatures (>80 °C) remains a critical challenge in industrial biotechnology owing to limited genetic tools and poor mechanistic understanding of microbial thermoadaptation. We aimed to develop a novel Geobacillus stearothermophilus strain with remarkable thermal resilience through an integrated approach combining adaptive laboratory evolution and rational genetic engineering. Progressive thermal adaptation (70–80 °C) followed by genome reduction generated a mutant (SL-1–80) with enhanced stability at 80 °C. Subsequent combinatorial overexpression of eight heat-associated genes (murD, cysM, grpE, groES, hsp33, hslO, hrcA, clpE) synergistically extended its survival to 85 °C. Genomic and transcriptomic analyses revealed a triple mechanism: (1) strategic deletion of transposable elements (IS5377/IS4/IS110) reduced genomic instability, (2) co-activation of chaperone systems (GroES-GrpE) and redox homeostasis enzymes (HslO—Hsp33) enhanced protein folding and oxidative stress resistance, and (3) metabolic plasticity (BglG and HTH-domain transcriptional repressor), motility optimization (FliY), and transcriptional reprogramming (Sigma-D, DUF47-family chaperone and HTH-domain transcriptional repressor) facilitated nutrient acquisition and motility-based environmental navigation under stress. Furthermore, we established the first high-efficiency electroporation protocol (104 transformants/µg DNA) for this genus, enabling ATP-enhanced heterologous protein expression under heat stress. This study provided a robust platform organism for high-temperature bioprocessing and a mechanistic blueprint for engineering microbial thermotolerance, addressing key limitations in applications such as microbial-enhanced oil recovery and industrial enzyme production.

Image, graphical abstract