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

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 (HslOHsp33) 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.