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subtilis. Following validation of the engineered transcriptional riboswitches using specialized reporter strains, our approach was implemented into a B. subtilis wild-type strain employing CRISPR-Cas9 genome editing. The resulting purine and riboflavin production strains were characterized at the level of gene expression, metabolite synthesis and growth, and a substantial enhancement was measured at each level. Moreover, applying our approach to deregulate the purine pathway of an industrial riboflavin overproducing strain with impaired growth led to an inc