Metabolic engineering of Methanothermobacter thermautotrophicus ΔH for recombinant acetoin production

Thermophilic methanogens of the genus Methanothermobacter are established biocatalysts in power-to-gas applications, converting H2 and CO2 into CH4 through the process of methanogenesis. Further expanding this platform for the bioproduction of value-added compounds (power-to-x) has the potential to increase the economic viability of such processes. This requires a genetic toolset that enables the controlled expression of recombinant pathways. Here, we report the fully autotrophic inducible recombinant bioproduction of acetoin from H2 and CO2 in Methanothermobacter thermautotrophicus {Delta}H. To facilitate inducible gene expression, we implemented an anhydrotetracycline (aTc)-inducible promoter system, expanding our available set of promoters. The aTc-inducible system enabled controlled expression of a codon-optimized acetoin-production operon comprising the acetolactate synthase- and acetolactate decarboxylase-encoding genes from Streptococcus thermophilus. Batch cultivation at 42{degrees}C demonstrated aTc-dependent acetoin formation, yielding up to 0.45 {+/-} 0.08 mM acetoin. Fed-batch bioreactor experiments confirmed growth-coupled, recombinant acetoin production, while eliminating the non-specific acetoin accumulation that we observed during non-growth phases in batch cultivation. Continuous cultivation in a chemostat resulted in stable acetoin production rates of 1.28 {+/-} 0.07 {micro}mol L-{superscript 1} h-{superscript 1} at 42{degrees}C. Elevated temperatures led to reduced acetoin production, suggesting diminished activity or thermal instability of the heterologous enzymes. This study demonstrates the feasibility of value-added bioproduction in Methanothermobacter and establishes an inducible expression system suitable for pathway engineering in thermophilic methanogens. Together with genome-scale modeling and emerging enzyme engineering strategies, these results lay the foundation for developing robust, CH4-co-producing power-to-x bioprocesses with Methanothermobacter species.