A systems-level view of metabolic impact of copper and methane valorization potential in Methylosinus trichosporium OB3b

Methanotrophs, which utilize methane as their sole carbon and energy source, are promising platforms for valorizing methane to industrially valuable products, such as bioplastics or biofuels. However, methanotrophic activity is highly affected by the availability of metals, and therefore, a greater understanding of these effects is important for efficient conversion of methane by methanotrophs. To address this, the impact of copper on methanotrophy was investigated at a systems level via the reconstruction of a genome-scale metabolic model (GEM) for Methylosinus trichosporium OB3b. The GEM was further integrated with transcriptomic data for improved functionality, which was then employed to identify metabolic engineering strategies for increased putrescine production. Despite these efforts, the GEM framework was not successful in significantly enhancing putrescine production. Therefore, the steady-state 13 C-metabolic flux analysis was conducted to estimate intracellular flux distributions and to validate the GEM functionality, revealing the indispensability of the ethylmalonyl-CoA cycle and inflexibility of flux utilization in central methane metabolism. These findings highlight key pathway constraints for methane valorization and expand the system-level understanding of copper-regulated methanotrophic metabolism. • A GEM of M. trichosporium OB3b reveals system-level effects of copper on metabolism. • Integration of transcriptomic data improves GEM functionality and biological relevance. • Limited flux variability in central metabolism hinders potential methane valorization. • The GEM predictions were validated using 13 C-metabolic flux analysis.