Establishment of tools for genetic modification of the thermophilic methanogenic archaeon Methanothermobacter thermautotrophicus deltaH

Abstract

The hydrogenotrophic methanogenic archaeon Methanothermobacter thermautotrophicus has been studied as a model microbe for methanogenesis and thermophilic microbial lifestyle in basic- and applied science for decades. Its short doubling times and robust growth behavior compared to other methanogenic archaea make M. thermautotrophicus a relatively accessible methanogenic archaeon. Combined with its high methane production rates, short lag phase after transfer, and thermophilic characteristics, M. thermautotrophicus is highly suitable for industrial scale biomethanation processes for energy storage and carbon recycling such as power-to-gas. In this study, we report the establishment of a reliable method for genetic modification of M. thermautotrophicus ΔH based on interdomain conjugation with E. coli S17-1 for DNA transfer. We developed the modular shuttle vector system pMVS (plasmid Methanothermobacter vector system) that facilitates transfer of genetic cargo into M. thermautotrophicus ΔH with exchangeable selectable markers and replicons for host and recipient microbe by the use of rare restriction enzyme recognition sites. In the first successful pMVS vector, a thermostable kanamycin resistance gene served as selectable marker against neomycin and the cryptic plasmid pME2001 from M. marburgensis as a replicon in M. thermautotrophicus ΔH. Additionally, we provide a platform to integrate heterologous DNA in the genomic DNA of M. thermautotrophicus ΔH via suicide vector constructs. We demonstrated site-specific genome integration at the mth58-mth61 (MTH_RS00275-290) fimbriae operons with a formate dehydrogenase operon (fdhZ-245) from M. thermautotrophicus Z-245 that enable M. thermautotrophicus ΔH to grow on formate, which qualifies fdhZ-245 as a prototrophy-based selectable marker. Furthermore, we integrated the thermostable neomycin resistance from the pMVS design. With that, we showed deletion of a target genomic sequence via double homologous recombination events by substitution with the neomycin resistance gene. Furthermore, the successful DNA transfer into M. thermautotrophicus enabled us to transport genetic cargo from E. coli to M. thermautotrophicus via the pMVS. With that, we proved heterologous gene expression of a thermostable β-galactosidase (bgaB) from Geobacillus stearothermophilus as a functional reporter gene on a pMVS vector in M. thermautotrophicus ΔH. For that, we performed quantitative in-vitro enzyme activity assays with o-Nitrophenyl-β-D-galactopyranosid (ONPG) as chromogenic substance and tested four different synthetic and native promoters, which resulted in significantly different expression levels of bgaB among the respective promoters.