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

Abstract

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.

Multi-tissue transcriptome analysis using hybrid-sequencing reveals potential genes and biological pathways associated with azadirachtin A biosynthesis in neem (Azadirachta indica) 

“…Alcohol at C3 is continuously oxidized into C3 ketone group by transcript/18725 and transcript/17679 and forms common compounds [73] isolated from Meliaceae family; these two unigenes expressed highest in root and fruit, respectively. Next important step involved in azadirachtin A is the formation of the furan ring; the two CYP450s catalyzing its formation were isolated from M. azedarach and C. sinensis [18]. Two transcripts (transcript/16971 and transcript/16742) in our study were found to be homologs of the two identi ed CYP450s and might produce melianone [74] from its precursor.…”

“…Among them, only transcript/14449 expressed higher in fruit. After phylogenetic analysis (Figure 3) of transcript/14449 with several characterized OSCs from other plants, transcript/14449 was grouped with AiOSC1 [18], a newly characterized OSC from neem, catalyzing the formation of tirucalla-7,24-dien-3β-ol; other unigenes were grouped with cycloartenol synthase. After DNA sequence analysis with AiOSC1( Figure S3), transcript/14449 is 100% identical to AiOSC1, which means that these two genes were the same gene.…”

“…All authors read and approved the nal manuscript. Homologs with * represent the identified gene in the previous report [18]. Q20 and Q30 on Illumina platform correspond to the predicted base call error rate of 1 % and 0.1%, respectively.…”

“…Among them, 3 transcripts encoding the complete AiOSC1 and homologs of MaCYP71CD2 and MaCYP71BQ5 were also found in our study. Unigene containing the complete open reading frame (ORF) encoding the homolog of MaCYP71BQ5 [18] was rst reported. Quantitative real-time PCR and full-length PCR cloning were used for verifying unigene expression level (Table S2) (Fragments per kilobase of transcript per million mapped reads (FPKM)) and transcript sequence accuracy.…”

Multi-tissue transcriptome analysis using hybrid-sequencing reveals potential genes and biological pathways associated with azadirachtin A biosynthesis in neem (Azadirachta indica)

“…The obtained candidates could be used as an important resource to investigate the catalysts responsible for essential biochemical reactions in azadirachtin A biosynthesis as well as triterpenoid metabolism in closely related species of neem. Homologs with * represent the identified gene in the previous report [18].…”