Static and Dynamic Regulation of Precursor Supply Pathways to Enhance Raspberry Ketone Synthesis from Glucose in <i>Escherichia coli</i>

Raspberry ketone (RK), a natural product derived from raspberry fruit, is commonly utilized as a flavoring agent in foods and as an active component for weight loss. Metabolic engineering has enabled microorganisms to produce RK more efficiently and cost-effectively. However, the biosynthesis of RK is hindered by an unbalanced synthetic pathway and a deficiency of precursors, including tyrosine and malonyl-CoA. In this study, we constructed and optimized the RK synthetic pathway in <i>Escherichia coli</i> using a static metabolic engineering strategy to enhance the biosynthesis of tyrosine from glucose, thereby achieving the <i>de novo</i> production of RK. Additionally, the synthetic and consumption pathways of malonyl-CoA were dynamically regulated by <i>p</i>-coumaric acid-responsive biosensor to balance the metabolic flux distribution between cell growth and RK biosynthesis. Following pathway optimization, the medium components and fermentation conditions were further refined, resulting in a significant increase in the RK titer to 415.56 mg/L. The optimized strain demonstrated a 32.4-fold increase in the RK titer while maintaining a comparable final OD₆₀₀ to the initial strain. Overall, the implemented static and dynamic regulatory strategies provide a novel approach for the efficient production of RK, taking into account cell viability and growth.