The triterpenes are one of the largest and most structurally diverse families of plant natural products.
Many triterpene derivatives have been shown to possess medicinally relevant biological activity. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic.
This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows.
Despite their immense diversity, triterpenes are all derived from a single linear precursor, 2,3-oxidosqualene.
Transient heterologous expression of biosynthetic enzymes in N. benthamiana can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products that are not naturally produced by this host. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana.
The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields.
After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment.
The platform is also extremely convenient for co-expression of different combinations of proteins, as this can be achieved facilely by the co-infiltration of different strains of A. tumefaciens, negating the need to build large multigene expression cassettes.
This process can be used to functionally characterise candidate enzymes and reconstruct the biosynthetic pathways of important naturally-occurring metabolites.
Equally, it can also be exploited in combinatorial biosynthetic approaches to produce novel triterpene products, a strategy that can result in libraries of structurally related analogs, allowing systematic exploration of the structure-activity relationships of biologically active lead compounds.