Disruption of ribosomal hibernation increases translational capacity in diverse microbial hosts

Ribosome hibernation factors (RHFs) inactivate ribosomes under stress to promote survival but limit biosynthetic capacity. While essential in fluctuating natural environments, this mechanism restricts productivity in laboratory and industrial settings. We found RHFs expression inversely correlates with growth rate and heterologous production, revealing a trade-off between stress adaptation and biosynthesis. Our physiological characterization showed broad fitness gains in Δrmf strains across 48 carbon sources, whereas ΔraiA mutants exhibited variable, condition-dependent effects. RHFs promoter activity varied with nutrient availability, displaying transient expression in rich media and sustained activity under poor carbon sources, highlighting their role in nutrient limitation. Deletion of raiA and rmf in E. coli increased translation, enhancing heterologous protein and metabolite production. Transcriptomics of ΔraiA revealed a shift from stress-responsive to growth-promoting programs. Extending this approach, RHF inactivation enhanced protein production in Pseudomonas putida and Saccharomyces cerevisiae, establishing ribosomal hibernation as a broadly engineerable target to enhance microbial production.