Limonoid biosynthesis 3: Functional characterization of crucial genes involved in neem limonoid biosynthesis. (April 2021)

• Record Type: Journal Article
• Authors: Pandreka, Avinash; Chaya, Patil S.; Kumar, Ashish; Aarthy, Thiagarayaselvam; Mulani, Fayaj A.; Bhagyashree, Date D.; B, Shilpashree H.; Jennifer, Cheruvathur; Ponnusamy, Sudha; Nagegowda, Dinesh; Thulasiram, Hirekodathakallu V.
• Is Part Of: Phytochemistry. Volume 184(2021)
• Publication Date: 2021-04
• Subjects: Azadirachta indica A. Juss. -- Meliaceae -- Transcriptome -- Limonoids -- Tirucalla-7, 24-dien-3β-ol -- Cytochrome P450 system; DMAPP Dimethylallyl pyrophosphate -- IPP Isopentenyl pyrophosphate -- MVA Mevalonate pathway -- MEP Methylerythritol phosphate pathway -- GPP Geranyl diphosphate -- FPP Farnesyl diphosphate -- GGPP Geranylgeranyl diphosphate -- FDS Farnesyl diphosphate synthase -- SQS Squalene synthase -- SQE Squalene epoxidase -- TTS Triterpene synthase -- CAS Cycloartenol synthase -- ORF Open Reading Frame; Botanical chemistry -- Periodicals; Biochemistry -- Periodicals; Botany -- Periodicals; Chimie végétale -- Périodiques; 572.2
• DOI: 10.1016/j.phytochem.2021.112669
• Languages: English
• ISSNs: 0031-9422

Abstract

Abstract: Neem ( Azadirachta indica L.) is well known for its medicinal, agricultural, and pesticidal applications since ages. The secondary metabolites, limonoids, confer these biological properties, wherein over 150 different limonoids have been reported from neem. To understand limonoid biosynthesis, we analyzed tissue-specific (kernel, pericarp, leaves, and flower) transcriptome that resulted in the identification of one farnesyl diphosphate synthase (AiFDS), one squalene synthase (AiSQS), three squalene epoxidases (AiSQE1, AiSQE2, and AiSQE3), two triterpene synthases (AiTTS1 and AiTTS2), cycloartenol synthase (AiCAS), two cytochrome P450 reductases, and ten cytochrome P450 systems. Comparative tissue-expression analysis indicated that AiFDS, AiSQS, AiSQE3, and AiTTS1 are expressed higher in the kernel than in the other tissues. Heterologously expressed recombinant AiTTS1 produced tirucalla-7, 24-dien-3β-ol as the sole product. Expression profile data, phylogeny with triterpene synthases from Meliaceae and Rutaceae families, real-time PCR of different tissues, and transient transformation revealed the involvement of tirucalla-7, 24-dien-3β-ol synthase (AiTTS1) in limonoid biosynthesis. Further, mutagenesis studies of AiTTS1 indicated that Y125 and F260 are probably involved in stabilization of dammarenyl cation. A 2.6-fold increase in production of tirucalla-7, 24-dien-3β-ol was observed when AiSQE1 was co-expressed with mutant AiTTS1 in a yeast system. Furthermore, we functionally characterized the highly expressed cytochrome P450 reductases and cycloartenol synthase. This study helps in further analysis and identification of genes involved in limonoid biosynthesis in Meliaceae/Rutaceae and their production in a metabolically tractable heterologous system. Graphical abstract: Image 1 Highlights: Tissue-specific transcriptome analysis elucidated limonoid biosynthesis in neem. Tirucalla-7, 24-dien-3β-ol synthase (AiTTS1), Squalene epoxidases, and CYP450 reductases were characterized. Transient transformation confirmed involvement of AiTTS1 in limonoid biosynthesis. Increased efficiency of AiTTS1 by rational mutagenesis. Heterologous production of tirucalla-7, 24-dien-3β-ol by co-expression of AiTTS1 and squalene epoxidase.