Heterologous Production of Forskolin in Tobacco ( <scp> <i>Nicotiana tabacum</i> </scp> ) via Glandular Trichome Specific Engineering and Metabolic Flux Redirection

Plants are promising bioreactor for the sustainable and scalable production of high-value natural bioactive compounds, because they can synthesise phytochemicals from CO₂, light, water and minerals through their innate photosynthetic carbon assimilation machinery. However, metabolic engineering in multicellular plants via stable transgene is challenged by the low accumulation of heterologous compounds. Forskolin is a labdane-type diterpenoid that accumulates in the roots of medicinal plant Coleus forskohlii. It activates the cyclic adenosine 3',5'-monophosphate (cAMP) signalling pathway and is used for the treatment of heart complications, respiratory disorders, high blood pressure, obesity and asthma. Here, we report the successful production of forskolin in transgenic tobacco (Nicotiana tabacum). Constitutive expression of six biosynthetic genes, including diterpene synthases (CfTPS2 and CfTPS3), cytochrome P450s (CYP76AH15, CYP76AH11, CYP76AH16) and acetyltransferase (CfACT1-8), results in low-level production of forskolin throughout the plant. In contrast, glandular trichome-specific expression of these genes leads to a significant increase in forskolin accumulation in the aerial parts of the plant. Further optimisation through CRISPR/Cas9 disruption of the biosynthetic pathway of endogenous diterpenoids α/β-cembratriene-diols and redirection of precursor flux towards forskolin biosynthesis improves its yield, reaching up to 21.26 μg/g fresh weight (206.33 μg/g dry weight) in the leaves of transgenic tobacco plants, surpassing the content in C. forskohlii roots. Our work not only provides a scalable and sustainable plant-based approach for forskolin production, but also represents a novel strategy of tissue- or organ-specific engineering and metabolic flux redirection to boost the heterologous production of high-value natural bioactive compounds in plants.