Metabolic engineering of Nicotiana benthamiana for production of curcuminoids

MAIN CONCLUSION: Nicotiana benthamiana plants transiently transformed with the C4 vector are capable to produce curcumin, demethoxycurcumin and bisdemethoxycurcumin in reasonable yields. Curcuminoids are valuable bioactive compounds whose heterologous production remains limited by precursor availability, cytotoxic intermediates and imbalanced metabolite ratios in microbial systems. In this study, we employed Nicotiana benthamiana as a platform for the transient expression of turmeric polyketide synthases, including diketide-CoA synthase (DCS) and curcumin synthases 1-3 (CURS1-3). Using Agrobacterium tumefaciens and a polycistronic expression vector (C4), we achieved the coordinated production of curcumin [187.7 ± 4.6 µg/g dry weight (DW)], demethoxycurcumin (41.8 ± 2.3 µg/g DW) and bisdemethoxycurcumin (31 ± 4.6 µg/g DW). Notably, the relative proportions of these metabolites closely matched those found in turmeric rhizomes. Metabolite identity was confirmed by chromatographic and spectroscopic analyses. These results demonstrate that plant-based transient expression systems can overcome key limitations of microbial platforms and enable the production of curcuminoids in physiologically relevant ratios.