New Methanol-Tolerant Microbe Boosts Bio-Refining

Abstract

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.

A research team, affiliated with UNIST has engineered a microbial strain capable of rapidly growing in high concentrations of methanol, marking a significant step forward in biorefinery technology. This breakthrough provides a foundational platform for sustainable biomanufacturing using microbial processes.

Led by Professor Donghyeok Kim in the School of Energy and Chemical Engineering at UNIST, the research employed adaptive laboratory evolution (ALE) to develop a methanol-tolerant microbial strain that can proliferate approximately 1.7 times faster than conventional strains under high methanol conditions. This advancement brings us closer to the practical application of biorefinery systems that convert methanol into valuable petrochemical derivatives.

The concept of a C1 biorefinery involves feeding microbes molecules containing a single carbon atom (C1), such as methanol, to produce plastics and chemicals traditionally derived from fossil fuels. Methanol is especially attractive due to its low cost, ease of storage, and transportability.

The newly developed strain demonstrates strong growth at 2.5% (v/v) methanol - a concentration at which typical microbes experience growth inhibition. Achieving rapid growth at such high substrate levels is crucial for making biorefinery processes economically viable.

The team applied adaptive evolution by gradually increasing methanol concentrations, selecting for microbes that could survive and adapt at each stage. Over successive generations, this process yielded "super strains" with enhanced resilience.

Genomic analysis revealed recurrent mutations in two key genes: metY, involved in methionine biosynthesis and detoxification of toxic byproducts, and kefB, which regulates cellular energy expenditure through potassium efflux. Functional validation confirmed that mutations in metY help suppress the formation of toxic compounds, while changes in kefB improve energy conservation-both mechanisms contributing to increased methanol tolerance.

Lead author Gyu Min Lee explained, "Identifying these genetic mutations provides a blueprint for designing high-performance microbial strains for methanol-based manufacturing. This enables us to develop engineered strains rapidly, bypassing lengthy evolution processes."

Professor Kim added, "This development could significantly lower production costs and increase yields in processes like bioplastic and organic acid manufacturing, making sustainable production more economically feasible."

The findings of this research have been published online in the Journal of Biological Engineering on January 12, 2026. This research was supported by the C1 Gas Refinery Program and the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science and ICT (MSIT), as well as the Circle Foundation.

Journal Reference

Gyu Min Lee, Khoi Nhat Pham, Ina Bang, et al., "Integrated genomic and transcriptomic Insights into methanol tolerance mechanisms in Methylobacterium extorquens AM1, identifying key targets for strain engineering," J. Biol. Eng., (2026).