| 초록 |
Methanol is an attractive one-carbon feedstock for sustainable biomanufacturing because of its abundance, cost-effectiveness, and industrial compatibility. However, its cytotoxicity limits its biotechnological applications in native methylotrophs such as Methylobacterium extorquens AM1. In this study, we developed AM1-derived strains capable of sustained growth under elevated methanol concentrations through adaptive laboratory evolution (ALE). From the evolved population, five representative strains were isolated, exhibiting up to a 1.68-fold increase in specific growth rates compared with those of the wild- type at 2.5% (v/v; 617.93 mM) methanol. Genomic analysis of the evolved strains revealed recurrent mutations in metY (O-acetyl-L-homoserine sulfhydrylase) and kefB (potassium efflux antiporter). Functional validation confirmed that these recurrent mutations improve methanol tolerance through distinct yet complementary mechanisms. The consistent emergence of mutations in metY and kefB across all strains implies strong convergent selection, highlighting their independent roles in a coordinated adaptive strategy. Specifically, the metY mutations are hypothesized to fine-tune enzyme activity to reduce toxic byproduct formation, while the loss-of-function kefB mutation likely conserves cellular energy. The largely additive nature of their combined effect underscores how these distinct adaptive mechanisms, optimization of methionine biosynthesis and energy conservation, independently contribute to the overall fitness improvement under methanol stress. To further elucidate methanol adaptation strategies, we performed an integrated genomic and transcriptomic analysis. Transcriptome profiling revealed 767 differentially expressed genes, indicating widespread transcriptional reprogramming. Notably, the key upregulated genes were involved mainly in central carbon metabolism, methionine biosynthesis, cellular defense responses such as oxidative stress mitigation, and nitrogen metabolism, as interpreted through DEG mapping onto metabolic pathways using a genome-scale metabolic model. Overall, this study highlights how coordinated genetic and transcriptional adaptations contribute to methanol tolerance in the AM1-derived evolved strains, providing systems-level insights. These strains represent promising platforms for methanol-based biomanufacturing, with the potential to improve microbial robustness and reduce stress-induced bottlenecks in industrial processes.
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GISCIENCE & REMOTE SENSING, v.63, no.1, 2026-12