Optimizing MatB-MatC-Dependent Malonyl-CoA Supply for Enhanced Raspberry Ketone Production in Escherichia coli.

Raspberry ketone (RK) is a valuable natural flavor compound, but its extraction from plants is inefficient because of its low natural abundance. Microbial production of RK offers a promising alternative; however, insufficient availability of malonyl-CoA can limit RK biosynthesis. In this study, we introduced a malonate-dependent malonyl-CoA supply module into an engineered Escherichia coli strain designed for de novo RK production through heterologous expression of malonyl-CoA synthetase MatB and malonate transporter MatC. In the presence of malonate, the introduction of the MatB-MatC module increased RK production by 2.3-fold. To optimize malonate supplementation, RK pathway metabolites, including intracellular acyl-CoA intermediates, were quantified by liquid chromatography-mass spectrometry, and the resulting metabolite profiles were analyzed by principal component analysis, hierarchical clustering, and correlation analysis. This study indicated that 50-mM malonate was optimal, yielding 24 mg/L RK during 96-deep-well plate cultivation. Fed-batch optimization increased RK production to 301 mg/L in a 100-mL jar fermenter, and scale-up cultivation in a 2-L jar fermenter produced 340 mg/L RK. In this study, optimizing MatB-MatC-dependent malonyl-CoA supply, combined with pathway-level metabolic profiling and controlled fed-batch cultivation, enhanced de novo RK production in E. coli.