Riboswitch-targeted improvement of lysine production in Bacillus and Priestia species

Lysine is an essential amino acid often limited in plant-based foods, making its enrichment in fermented foods a key nutritional goal. Bacillus and Priestia species are natural lysine producers, but their biosynthetic capacity is constrained by riboswitch regulation of the aspartokinase gene lysC. In this study, we employed mutagenesis using the lysine analogue aminoethylcysteine (AEC) to generate lysine-overproducing strains. AEC-resistant mutants carried distinct mutations in the lysC riboswitch and in lysine biosynthetic and transporter genes. Several mutants showed significantly increased lysine production in defined media. One riboswitch mutant produced over 150-fold more free lysine than its wild type in defined media and threefold during oat fermentation, establishing proof of concept for lysine enrichment in food matrices. Structural predictions revealed that lysC riboswitch mutations occurred near the lysine binding pocket, suggesting altered ligand-binding dynamics. These findings highlight riboswitch-targeted mutagenesis as a promising non-recombinant strategy to enhance lysine production in food fermentations.