A robust genetic toolbox for fine-tuning gene expression in the CO₂-Fixing methanogenic archaeon Methanococcus...

Libraries of well-characterized genetic elements for fine-tuning gene expression are essential for biological and biotechnological research and applications. The fast-growing and genetically tractable methanogen, Methanococcus maripaludis, is a promising host organism for biotechnological conversion of carbon dioxide and renewable hydrogen into fuels and value-added products, as well as fundamental biological studies of archaea. However, the lack of molecular tools for gene expression has hindered its application as a workhorse to fine-tune gene and metabolic pathway expressions. In this study, we developed a genetic toolbox, including libraries of promoters, ribosome binding sites (RBS), and neutral sites for chromosomal integration, to facilitate precise gene expression in M. maripaludis. We generated a promoter library consisting of 81 constitutive promoters with expression strengths spanning a ∼10⁴-fold dynamic range. Importantly, we identified a base composition rule for strong archaeal promoters and successfully remodeled weak promoters, enhancing their activities by up to 120-fold. We also established an RBS library containing 42 diverse RBS sequences with translation strengths covering a ∼100-fold dynamic range. Additionally, we identified eight neutral sites and developed a one-step, Cas9-based marker-less knock-in approach for chromosomal integration. We successfully applied the characterized promoter and RBS elements to significantly improve recombinant protein expression by 41-fold and modulate essential gene expression to generate corresponding physiological changes in M. maripaludis. Therefore, this work establishes a solid foundation for utilizing this autotrophic methanogen as an ideal workhorse for archaeal biology and biotechnological studies and applications.