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 protein production, revealing a trade-off between stress adaptation and biosynthesis. Our physiological characterization showed broad fitness gains in Δ rmf strains across 95 carbon sources, whereas Δ raiA mutants exhibited variable, condition-dependent effects, indicating distinct roles. RHFs promoter activity varied with nutrient availability, displaying transient expression in rich media and sustained activity under poor carbon sources, highlighting ribosomal hibernation’s role in nutrient limitation. Deletion of raiA and rmf in E. coli increased translation, boosting heterologous protein and metabolite yields. Transcriptomics of Δ raiA revealed a shift from stress-responsive to growth-promoting programs. Extending this approach, RHFs inactivation enhanced protein production in Pseudomonas putida and Saccharomyces cerevisiae , establishing ribosomal hibernation as a broadly engineerable target to enhance microbial production.