Promoter characterization for metabolic engineering in Methanococcus maripaludis

Methanococcus maripaludis is a hydrogenotrophic methanogen which has become a model organism for archaea and for the study of methanogenesis. New genetic and genomic tools hold promise to convert M. maripaludis into a host organism for synthetic biology and metabolic engineering. However, an extensive characterization of suitable promoters in this organism for their use in metabolic engineering has been lacking. In this study, the strength of 25 promoter sequences was quantified by use of a β-glucuronidase reporter gene assay, establishing it as an adequate method for this purpose. PglnA seemed to evade the control exerted by the native transcriptional regulator NrpR and thus became the strongest promoter of all tested. Pmtr, Pmcr, , Pmcr_JJ and Ppor_JJ could also be regarded as strong promoters in this organism. On the other hand, inclusion of a putative transcription factor downstream of the eha operon increased the strength of Peha and Peha_JJ, suggesting its role as the activator of the operon. Overall, these results provide valuable information for the implementation of genetic, metabolic and promoter engineering in M. maripaludis. Genetic manipulation of M. maripaludis was done via a recently developed CRISPR/Cas toolbox, serving as an example of its efficiency.