Establishing the fluorescence-activating and absorption-shifting tag as a fluorescent reporter protein in Methanothermobacter thermautotrophicus ΔH

The thermophilic methanogen Methanothermobacter thermautotrophicus ΔH is a model microbe for hydrogenotrophic methanogenesis and an emerging platform host for metabolic engineering. Despite recent advances in its genetic accessibility, the available molecular toolbox lacks fluorescent reporter proteins that are suitable for anaerobic and thermophilic conditions. Here, we established the fluorescence-activating and absorption-shifting tag (FAST) as a reporter protein in M. thermautotrophicus . We expressed codon-optimized variants of FAST by applying established genetic tools, and evaluated the performance for two temperatures and three fluorogens. We demonstrated that FAST is functional in M. thermautotrophicus but exhibits temperature-dependent instability, which is more pronounced at 60°C compared to 50°C. Among the tested fluorogens, ^TF Lime and ^TF Amber yielded comparable fluorescence intensities, while ^TF Coral resulted in significantly lower fluorescence intensity. Exploiting the partial thermolability of FAST, we characterized the dynamic expression profiles of several promoters, which revealed growth phase-dependent regulation patterns. Our findings challenge previous assumptions of constitutive expression for several promoters. Notably, we identified distinct expression patterns for promoters that are associated with methanogenesis and energy-converting hydrogenases. Our results establish FAST as a versatile fluorescent reporter for thermophilic methanogens and provide new insights into promoter regulation in M. thermautotrophicus . This work expands the genetic toolbox for this microbe and lays the foundation for advanced studies in archaeal cell biology and biotechnology.