Limonoids are natural products made by plants belonging to the Meliaceae (Mahogany) and Rutaceae (Citrus) families. They are well known for their insecticidal activity, contribution to bitterness in citrus fruits, and potential pharmaceutical properties. The best known limonoid insecticide is azadirachtin, produced by the neem tree (Azadirachta indica). Despite intensive investigation of limonoids over the last half century, the route of limonoid biosynthesis remains unknown. Limonoids are classified as tetranortriterpenes because the prototypical 26-carbon limonoid scaffold is postulated to be formed from a 30-carbon triterpene scaffold by loss of 4 carbons with associated furan ring formation, by an as yet unknown mechanism. Here we have mined genome and transcriptome sequence resources for 3 diverse limonoid-producing species (A. indica, Melia azedarach, and Citrus sinensis) to elucidate the early steps in limonoid biosynthesis. We identify an oxidosqualene cyclase able to produce the potential 30-carbon triterpene scaffold precursor tirucalla-7,24-dien-3β-ol from each of the 3 species. We further identify coexpressed cytochrome P450 enzymes from M. azedarach (MaCYP71CD2 and MaCYP71BQ5) and C. sinensis (CsCYP71CD1 and CsCYP71BQ4) that are capable of 3 oxidations of tirucalla-7,24-dien-3β-ol, resulting in spontaneous hemiacetal ring formation and the production of the protolimonoid melianol. Our work reports the characterization of protolimonoid biosynthetic enzymes from different plant species and supports the notion of pathway conservation between both plant families. It further paves the way for engineering crop plants with enhanced insect resistance and producing high-value limonoids for pharmaceutical and other applications by expression in heterologous hosts.

Momilactones from rice have allelopathic activity, the ability to inhibit growth of competing plants. Transferring momilactone production to other crops is a potential approach to combat weeds, yet a complete biosynthetic pathway remains elusive. Here, we address this challenge through rapid gene screening in N. benthamiana , a heterologous plant host. To do so, we solved a central problem with this technique: diminishing yields remain a bottleneck for multi-step pathways. We increased intermediate and product titers by re-routing diterpene biosynthesis from the chloroplast to the cytosolic, high-flux mevalonate pathway. This enabled the discovery and reconstitution of a complete route to momilactones (>10-fold yield improvement versus rice). Pure momilactone B isolated from N. benthamiana inhibits germination and root growth in Arabidopsis , validating allelopathic activity. We demonstrate the broad utility of this approach by applying it to forskolin, a hedgehog inhibitor, and taxadiene, an intermediate in taxol biosynthesis (~10-fold improvement versus chloroplast expression).

While RNA structures have been extensively characterized in vitro, very few techniques exist to probe RNA structures inside cells. Here, we have exploited mechanisms of post-transcriptional regulation to synthesize fluorescence-based probes that assay RNA structures in vivo. Our probing system involves the co-expression of two constructs: (i) a target RNA and (ii) a reporter containing a probe complementary to a region in the target RNA attached to an RBS-sequestering hairpin and fused to a sequence encoding the green fluorescent protein (GFP). When a region of the target RNA is accessible, the area can interact with its complementary probe, resulting in fluorescence. By using this system, we observed varied patterns of structural accessibility along the length of the Tetrahymena group I intron. We performed in vivo DMS footprinting which, along with previous footprinting studies, helped to explain our probing results. Additionally, this novel approach represents a valuable tool to differentiate between RNA variants and to detect structural changes caused by subtle mutations. Our results capture some differences from traditional footprinting assays that could suggest that probing in vivo via oligonucleotide hybridization facilitates the detection of folding intermediates. Importantly, our data indicate that intracellular oligonucleotide probing can be a powerful complement to existing RNA structural probing methods.

Triterpenes with complex scaffold modifications are widespread in the plant kingdom. Limonoids are an exemplary family that are responsible for the bitter taste in citrus (e.g., limonin) and the active constituents of neem oil, a widely used bioinsecticide (e.g., azadirachtin). Despite the commercial value of limonoids, a complete biosynthetic route has not been described. We report the discovery of 22 enzymes, including a pair of neofunctionalized sterol isomerases, that catalyze 12 distinct reactions in the total biosynthesis of kihadalactone A and azadirone, products that bear the signature limonoid furan. These results enable access to valuable limonoids and provide a template for discovery and reconstitution of triterpene biosynthetic pathways in plants that require multiple skeletal rearrangements and oxidations.

Triterpenes with complex scaffold modifications are widespread in plants yet little is known regarding biosynthesis. Limonoids are an exemplary family that includes the bitter taste in citrus (e.g., limonin) and the active constituents in neem oil, a widely used bioinsecticide (e.g., azadirachtin). Despite limonoid commercial value, a complete biosynthetic route has not been described. Here, we report the discovery of 22 enzymes that catalyze 12 unique reactions including the 4-carbon scission and furan installation that are a signature of the limonoid family and a pair of sterol isomerases previously unknown in plants. This gene set is then used for the total biosynthesis of kihadalactone A and azadirone in a heterologous plant. These results enable access to valuable limonoids and provide a template for discovery and reconstitution of triterpene biosynthetic pathways in plants that require multiple skeletal rearrangements and oxidations.