(509a) Quantitative Analysis of Antisense RNA-Mediated Inhibition and Application in the Fine-Tuning of Biochemical Production (Industry Candidate)

One major challenge for metabolic engineering is achieving the balance between cellular growth and production. Engineering efforts have thus combined gene knockout and knockdown approaches in static or dynamic manners. Genetic knockdown often fulfilled by RNA-based tools (such as asRNA, sRNA or CRISPRi), while most applications are limited to on-off regulations. Experimental and modeling results have suggested the need to fine tune the metabolic flux to further improve strains performance. To advance our knowledge in such direction, we systematically studied the effects of antisense RNA-based knockdown and characterized key factors contributing to different inhibitory levels. We then sought to establish a thermodynamic model to describe and predict such phenomenon based on competitive binding and RNA-RNA interactions. A fine-tuning tool for cellular activity was developed based on this quantifiable inhibition strategy. Finally, we use this tool to fine adjust the biosynthesis pathway flux for 4-hydroxycoumarin by quantifiable knockdown of multiple targets. We believe the develop tool provides an extra layer of control in modifying living systems and is broadly compatible with metabolic engineering strategies.