Expanding the genetic toolbox for Methanothermobacter thermautotrophicus ΔH

Methanothermobacter spp. are thermophilic, chemolithotrophic methanogens with a long history in basic research as model microbes for hydrogenotrophic methanogenesis. There is also industrial interest in Methanothermobacter spp. as biocatalysts in the power-to-gas process in which hydrogen that is produced with electricity from renewable resources is combined with carbon dioxide to produce renewable methane. The lack of genetic tools has hindered further investigations of Methanothermobacter spp. and the expansion of the power-to-gas process to produce value-added products. This only changed recently with the development of a genetic system for Methanothermobacter thermautotrophicus ΔH in our lab. After successfully establishing the genetic system, we focus on expanding the genetic toolbox. For example, we established the formate dehydrogenase (fdh) operon from M. thermautotrophicus Z-245 as an additional selection marker. Here, an alternative transformation method to the already established conjugation protocol is presented. For this, we adapted and optimized an electroporation protocol for the use with M. thermautotrophicus ΔH. Furthermore, we present results on applying a thermostable Cas9 from a Geobacillus strain as a tool for genome editing by deleting the gene encoding uracil phosphoribosyl transferase (upt), a possible counterselection marker. We discuss which adaptations to the transformation protocol and cell treatments were required to achieve a clean deletion. The presented additions to the genetic toolbox for M. thermautotrophicus ΔH can be used to advance industrial applications and fundamental research.