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

Abstract

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.

Keywords

Geobacillus stearothermophilus / Bacterial thermotolerance / Adaptive laboratory evolution / Engineering microorganism

Data Availability Statement

All relevant data supporting the findings of this study are available in this manuscript and the supplementary materials.

Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Given his role as Editor-in-Chief, Dr. Youming Zhang, had no involvement in the peer-review of this article and has no access to information regarding its peer-review. Full responsibility for the editorial process for this article was delegated to Dr. Shengbiao Hu.

CRediT authorship contribution statement

Jie Cui: Writing - original draft, Data curation, Conceptualization. Caifeng Li: Data curation. Gongze Cao: Methodology. Yuxia Wu: Resources. Shouying Xu: Project administration. Youming Zhang: Writing - review & editing, Supervision. Xiaoying Bian: Writing - review & editing, Supervision. Qiang Tu: Writing - review & editing, Supervision. Wentao Zheng: Writing - review & editing, Supervision, Project administration.

Acknowledgments

This work was supported by the National Key R&D Program of China (grant number 2023YFC3402003), the National Natural Science Foundation of Shandong province (grant number ZR2023YQ028), Taishan Scholar Program of Shandong Province in China (grant number tsqn202312007), Guangdong Basic and Applied Basic Research Foundation (grant number 2022A1515110795), the Recruitment Program of Global Experts (grant number 1000 Plan), the Program of Introducing Talents of Discipline to Universities (grant number B16030), and SKLMT Frontiers and Challenges Project of Shandong University. We thank Haiyan Yu, Nannan Dong and Xiangmei Ren of the Core Facilities for Life and Environmental Sciences, State Key laboratory of Microbial Technology of Shandong University for samples testing.

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