Stressostat cultivation of Lactococcus lactis improves lactate stress resistance through mutations in RNA polymerase

Adaptive laboratory evolution is used to improve the phenotypes of microorganisms and to characterise the mechanisms underlying resistance against complex growth inhibition. Here we focused on lactic acid bacteria (LAB) as starter cultures for food fermentations. Production of LAB starter cultures is challenging due to growth inhibition by organic acids, mainly lactate, produced during fermentation. By utilising stressostat cultivation we generated Lactococcus lactis isolates with enhanced lactate resistance. Using a combination of (meta)genomics, proteomics and pH-controlled batch fermentations, we deciphered the lactate resistance mechanisms of these L. lactis isolates. Proteome responses of L. lactis , combined with similar growth inhibition at high salt, suggest that high lactate mainly causes osmotic stress. We identified RNA polymerase (RNAP) mutations in subunits β ( rpoB ) and β’ ( rpoC ) as key mutations, causing pleiotropic effects in the proteome. These proteome adaptations are linked to enhanced lactate resistance, particularly the resistance to hyperosmotic stress without glycine-betaine supplementation, likely by altering cross-linking of the peptidoglycan in the cell envelope via downregulation of MurE. The proteome changes indicate that lactate might (indirectly) cause oxidative stress. Combined, our study shows that RNAP mutations enhanced lactate resistance through pleotropic effects in the proteome that changed L. lactis responses against multiple stresses. Highlights Lactate resistant variants were isolated from stressostat cultivations Metagenomics revealed evolutionary trajectory during stressostat cultivation RNAP mutations were identified as key drivers for improved lactate resistance RNAP mutations altered the proteome, enhancing the osmotic stress resistance