Riboswitches as targets for metabolic engineering in Bacillus subtilis

Many metabolic pathways in bacteria are modulated by metabolite-sensing riboswitches, which regulate gene expression at the level of transcription elongation or translation initiation. Riboswitches represent promising targets to modulate expression of genes and operons relevant for the biotechnological production of commercially relevant compounds. In Firmicutes, approximately 70% of all putative and validated riboswitches (are predicted to) act exclusively at the transcriptional level using a termination-antitermination mechanism. In a first attempt to interfere with purine-sensing riboswitches and deregulate purine metabolism in Bacillus subtilis, a set of synthetic small RNAs (sRNAs) targeting the purine-sensing aptamers were designed to impair ligand binding using rational design combined with in silico evolution. However, the designed sRNAs did not show any activity in vivo on the riboswitch controlling purine biosynthesis (pur operon riboswitch). The effect of the antisense RNA (asRNA) perfectly complementary to the aptamer of the pur operon riboswitch was also tested; The asRNA did not affect negatively expression of a riboswitch-regulated lacZ gene, yet similarly to the partially complementary sRNAs, the asRNA did not impair the downregulation exerted by the riboswitch in the presence of ligand. Finally, expression of the small RNAs in B. subtiliswas quantified, and the kinetic limitations for their hybridization with the aptamer and their competition with the ligand are discussed. A second metabolic engineering strategy based on editing the genome of B. subtilis with regard to transcriptional riboswitches was investigated. Removal of the riboswitches that control purine biosynthesis and riboflavin biosynthesis in B. subtilis led to auxotrophic strains. As an alternative, a rational approach was developed for engineering transcriptional riboswitches independently from the availability of their 3D structures. This approach consists in the identification and deletion of a key nucleotide sequence exclusively involved in transcription termination without affecting formation of other secondary and tertiary structures potentially involved in other roles. To demonstrate the efficacy of the approach, it was applied to derepress the purine and the riboflavin biosynthetic pathways in B. subtilis. Following the proof of concept using specialized reporter strains, the approach was implemented into a B. subtilis wild-type strain employing CRISPR-Cas genome editing. The CRISPRCas9 system displayed an efficiency of 61% in editing the genome, and the resulting purine and riboflavin production strains were characterized at the level of gene expression, metabolite synthesis, and growth. With a substantial enhancement observed at each level, the strategy established here represents a powerful tool for deregulating pathways modulated by transcriptional riboswitches. Finally, applying this strategy to derepress the purine pathway of an industrial riboflavin overproducing strain, with impaired growth, led to an increase in biomass by 53% and resulted in an enhanced total production of riboflavin in the culture