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Identification of key enzymes responsible for protolimonoid biosynthesis in plants: Opening the door to azadirachtin production

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Significance Limonoids are natural products made by members of the Meliaceae and Rutaceae families. Some limonoids (e.g., azadirachtin) are toxic to insects yet harmless to mammals. The use of limonoids… Click to show full abstract

Significance Limonoids are natural products made by members of the Meliaceae and Rutaceae families. Some limonoids (e.g., azadirachtin) are toxic to insects yet harmless to mammals. The use of limonoids in crop protection and other applications currently depends on extraction from limonoid-producing plants. Metabolic engineering offers opportunities to generate crop plants with enhanced insect resistance and also to produce high-value limonoids (e.g., for pharmaceutical use) by expression in heterologous hosts. However, to achieve this the enzymes responsible for limonoid biosynthesis must first be characterized. Here we identify 3 conserved enzymes responsible for the biosynthesis of the protolimonoid melianol, a precursor to limonoids, from Melia azedarach and Citrus sinensis, so paving the way for limonoid metabolic engineering and diversification. 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.

Keywords: biosynthesis; production; enzymes responsible; azadirachtin; citrus; limonoid biosynthesis

Journal Title: Proceedings of the National Academy of Sciences of the United States of America
Year Published: 2019

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