Chemical Biology Unit, Division of Organic Chemistry, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, India; CSIR-Institute of Genomics and Integrative Biology, Mall Road, New Delhi, 110007, India.
Chemical Biology Unit, Division of Organic Chemistry, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, India.
Phytochemistry. 2021 Apr;184:112669. doi: 10.1016/j.phytochem.2021.112669. Epub 2021 Jan 29.
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.
印楝（Azadirachta indica L.）因其药用、农业和农药应用而闻名于世。次级代谢产物柠檬素赋予了这些生物特性，从印楝中已经报道了超过 150 种不同的柠檬素。为了了解柠檬素的生物合成，我们分析了组织特异性（核、果皮、叶和花）转录组，结果鉴定了一个法呢基二磷酸合酶（AiFDS）、一个角鲨烯合酶（AiSQS）、三个角鲨烯环氧化酶（AiSQE1、AiSQE2 和 AiSQE3）、两个三萜合成酶（AiTTS1 和 AiTTS2）、环阿屯醇合酶（AiCAS）、两个细胞色素 P450 还原酶和十个细胞色素 P450 系统。比较组织表达分析表明，AiFDS、AiSQS、AiSQE3 和 AiTTS1 在核中表达高于其他组织。异源表达重组 AiTTS1 产生了作为唯一产物的羽扇豆-7,24-二烯-3β-醇。不同组织的表达谱数据、与金缕梅科和芸香科三萜合成酶的系统发育、实时 PCR 和瞬时转化揭示了羽扇豆-7,24-二烯-3β-醇合酶（AiTTS1）在柠檬素生物合成中的参与。此外，AiTTS1 的突变研究表明，Y125 和 F260 可能参与稳定达玛烯阳离子。当 AiSQE1 与突变体 AiTTS1 在酵母系统中共表达时，观察到羽扇豆-7,24-二烯-3β-醇的产量增加了 2.6 倍。此外，我们还对高度表达的细胞色素 P450 还原酶和环阿屯醇合酶进行了功能表征。这项研究有助于进一步分析和鉴定金缕梅科/芸香科中参与柠檬素生物合成的基因，并在代谢上可处理的异源系统中生产它们。
