LexA deficiency confers enhanced thermotolerance and glycogen accumulation in Methylomicrobium buryatense

Systematic mining of heat stress factors is pivotal for constructing robust microbial cell factories. However, the genetic basis underlying thermotolerance remains poorly understood in C1 gas-utilizing chassis. Here, we identified LexA as a key regulator governing thermotolerance and glycogen accumulation in the methanotroph Methylotuvimicrobium buryatense 5GB1C. Through ultraviolet mutagenesis coupled with adaptive laboratory evolution, we developed a thermotolerant evolved strain 5C27. Whole-genome resequencing and transcriptomic analysis revealed global transcriptional reprogramming underlying the heat tolerance phenotype. Notably, LexA was sharply downregulated at 40 °C and identified as a core regulatory factor for thermoadaptation. Transcriptomic analysis indicated that the acquired thermotolerance arises from coordinated regulation of oxidative stress response, energy metabolism, and cellular homeostasis. Subsequent validation experiments demonstrated that lexA deficiency simultaneously conferred enhanced thermotolerance and redirected intracellular carbon flux, resulting in increased glycogen production that exceeded wild-type levels even at the optimal growth temperature of 30 °C. Collectively, our work demonstrated for the first time that LexA plays a critical regulatory role in thermotolerance of methanotrophs. This discovery also provides a foundation for advancing the development of thermotolerant cell factories and promotes the deployment of C1-gas industrial biomanufacturing.