Tonoplast‐Localized ABCC Transporters Enhance Heterologous Production of the Antidiabetic Montbretin A and Its Analogues in Nicotiana benthamiana

The plant-derived specialized metabolite montbretin A (MbA) is under development as an improved treatment for Type 2 diabetes, a worldwide epidemic. MbA is an acylated flavonol glycoside so far only found in small amounts in montbretia (Crocosmia × crocosmiiflora) corms. The compound's structural complexity and low natural abundance present major challenges for large-scale production. To enable the development of a scalable MbA production system, we have previously engineered Nicotiana benthamiana to produce MbA. In addition to low levels of MbA, N. benthamiana also produces the pharmaceutically inactive analogues montbretin B and montbretin C, differing from MbA only in their acyl moieties. Follow-up research focusing on precursor engineering has improved MbA yield, but further yield optimization is required to establish N. benthamiana as a scalable MbA production system. Here, we report evidence that transport likely constrains heterologous montbretin production in N. benthamiana and that transporters can be leveraged as bioengineering tools to enhance yield. Specifically, co-expression of tonoplast localized CcABCC4 transporters with montbretin biosynthetic genes in N. benthamiana increased montbretin levels, including those of MbA. Yield increase is likely due to vacuolar sequestration and relies on the presence of an acyl group on the molecule. Eight out of fifteen ABCC4/14 transporters from plant species not known to produce montbretins also enhanced montbretin accumulation in N. benthamiana. These observations suggest that ABCC transporters might be versatile tools for bioengineering.