Characterisation of protolimonoid biosynthesis in plants: a key step towards engineering limonoids for crop protection.

The limonoids are a diverse class of plant natural product produced by the Meliaceae (Mahogany) and Rutaceae (Citrus) families. Limonoids are recognisable by their furan-containing tetracyclic triterpene structures, a number of which have human-usable biological activities. For instance, azadirachtin (from Azadirachta indica (neem)) is used to protect crops from insect pests. The current-use of limonoids is reliant on their extraction from plant materials, which has limited the extent of their use. Metabolic engineering could provide an alternative supply of limonoids. However, at the commencement of this project, the metabolic engineering of limonoids was not a possibility as the enzymes responsible for even the first step in their biosynthesis remained unknown. Here, the initiation of limonoid biosynthesis has been characterised in three diverse limonoid-producing species, A. indica, Melia azedarach (chinaberry) and Citrus sinensis (sweet orange). Three oxidosqualene cyclases (AiOSC1, MaOSC1 and CsOSC1) have been characterised as functional tirucalla-7,24-dien-3β-ol synthases which initiate limonoid biosynthesis. Further, two cytochrome P450s (MaCYP71CD2 and MaCYP71BQ5) from M. azedarach have been shown to collectively introduce three oxidations onto the side-chain of this scaffold to produce the protolimonoid melianol, a precursor to limonoids. The availability of Nagoya Protocol compliant M. azedarach plants has allowed the limonoid content of this species to be profiled and enabled the generation of a pseudochromosome-level genome assembly with accompanying RNA-Seq datasets. Utilising these resources a further four limonoid biosynthetic enzymes have be identified. Two of these enzymes (MaCYP88A108 and MaIsom1-I) are predicted to convert the internal scaffold of melianol to one of a mature limonoid and the other two enzymes (MaSDR1 and MaBAHD1) are thought to decorate the resultant structure. This biosynthetic knowledge will provide a basis for future metabolic engineering of limonoids for crop protection and has already enabled Nicotiana benthamiana-based investigations into the structure-activity relationships of limonoids