02340nas a2200205 4500008004100000022001400041245010400055210006900159260009000228300002600318490000600344520149100350100003101841700002501872700002701897700002201924700001901946700003701965856013202002 2015 eng d a2045-232200aFunctional characterization of novel sesquiterpene synthases from Indian sandalwood, santalum album0 aFunctional characterization of novel sesquiterpene synthases fro aMACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLANDbNATURE PUBLISHING GROUPcMAY aArticle Number: 100950 v53 a

Indian Sandalwood, Santalum album L. is highly valued for its fragrant heartwood oil and is dominated by a blend of sesquiterpenes. Sesquiterpenes are formed through cyclization of farnesyl diphosphate (FPP), catalyzed by metal dependent terpene cyclases. This report describes the cloning and functional characterization of five genes, which encode two sesquisabinene synthases (SaSQS1, SaSQS2), bisabolene synthase (SaBS), santalene synthase (SaSS) and farnesyl diphosphate synthase (SaFDS) using the transcriptome sequencing of S. album. Using Illumina next generation sequencing, 33.32 million high quality raw reads were generated, which were assembled into 84,094 unigenes with an average length of 494.17 bp. Based on the transcriptome sequencing, five sesquiterpene synthases SaFDS, SaSQS1, SaSQS2, SaBS and SaSS involved in the biosynthesis of FPP, sesquisabinene, beta-bisabolene and santalenes, respectively, were cloned and functionally characterized. Novel sesquiterpene synthases (SaSQS1 and SaSQS2) were characterized as isoforms of sesquisabinene synthase with varying kinetic parameters and expression levels. Furthermore, the feasibility of microbial production of sesquisabinene from both the unigenes, SaSQS1 and SaSQS2 in non-optimized bacterial cell for the preparative scale production of sesquisabinene has been demonstrated. These results may pave the way for in vivo production of sandalwood sesquiterpenes in genetically tractable heterologous systems.

1 aSrivastava, Prabhakar, Lal1 aDaramwar, Pankaj, P.1 aKrithika, Ramakrishnan1 aPandreka, Avinash1 aShankar, Shiva1 aThulasiram, Hirekodathakallu, V. uhttps://library.ncl.res.in/content/functional-characterization-novel-sesquiterpene-synthases-indian-sandalwood-santalum-album-103334nas a2200277 4500008004100000022001400041245014500055210006900200260008100269300002400350490000700374520225400381653002302635653002702658653001802685653001802703100002202721700002702743700001902770700002202789700002702811700002202838700002902860700003702889856013002926 2015 eng d a1471-222900aTriterpenoid profiling and functional characterization of the initial genes involved in isoprenoid biosynthesis in neem (Azadirachta indica)0 aTriterpenoid profiling and functional characterization of the in a236 GRAYS INN RD, FLOOR 6, LONDON WC1X 8HL, ENGLANDbBIOMED CENTRAL LTDcSEP aArticle Number: 2140 v153 a

Background: Neem tree (Azadirachta indica) is one of the richest sources of skeletally diverse triterpenoids and they are well-known for their broad-spectrum pharmacological and insecticidal properties. However, the abundance of Neem triterpenoids varies among the tissues. Here, we delineate quantitative profiling of fifteen major triterpenoids across various tissues including developmental stages of kernel and pericarp, flower, leaf, stem and bark using UPLC-ESI (+)-HRMS based profiling. Transcriptome analysis was used to identify the initial genes involved in isoprenoid biosynthesis. Based on transcriptome analysis, two short-chain prenyltransferases and squalene synthase (AiSQS) were cloned and functionally characterized. Results: Quantitative profiling revealed differential abundance of both total and individual triterpenoid content across various tissues. RNA from tissues with high triterpenoid content (fruit, flower and leaf) were pooled to generate 79.08 million paired-end reads using Illumina GA.. platform. 41,140 transcripts were generated by d e novo assembly. Transcriptome annotation led to the identification of the putative genes involved in isoprenoid biosynthesis. Two short-chain prenyltransferases, geranyl diphosphate synthase (AiGDS) and farnesyl diphosphate synthase (AiFDS) and squalene synthase (AiSQS) were cloned and functionally characterized using transcriptome data. RT-PCR studies indicated five-fold and ten-fold higher relative expression level of AiSQS in fruits as compared to leaves and flowers, respectively. Conclusions: Triterpenoid profiling indicated that there is tissue specific variation in their abundance. The mature seed kernel and initial stages of pericarp were found to contain the highest amount of limonoids. Furthermore, a wide diversity of triterpenoids, especially C-seco triterpenoids were observed in kernel as compared to the other tissues. Pericarp, flower and leaf contained mainly ring-intact triterpenoids. The initial genes such as AiGDS, AiFDS and AiSQS involved in the isoprenoids biosynthesis have been functionally characterized. The expression levels of AiFDS and AiSQS were found to be in correlation with the total triterpenoid content in individual tissues.

10aAzadirachta indica10aQuantitative profiling10aTranscriptome10aTriterpenoids1 aPandreka, Avinash1 aDandekar, Devdutta, S.1 aHaldar, Saikat1 aUttara, Vairagkar1 aVijayshree, Shinde, G.1 aMulani, Fayaj, A.1 aAarthy, Thiagarayaselvam1 aThulasiram, Hirekodathakallu, V. uhttps://library.ncl.res.in/content/triterpenoid-profiling-and-functional-characterization-initial-genes-involved-isoprenoid-103475nas a2200205 4500008004100000020001400041245017500055210006900230260000800299490000700307520265300314100002902967700002202996700002203018700001803040700002703058700001903085700003703104856012803141 2018 eng d a1471-222900a Tracing the biosynthetic origin of limonoids and their functional groups through stable isotope labeling and inhibition in neem tree (Azadirachta indica) cell suspension0 aTracing the biosynthetic origin of limonoids and their functiona cOCT0 v183 aBackground Neem tree serves as a cornucopia for triterpenoids called limonoids that are of profound interest to humans due to their diverse biological activities. However, the biosynthetic pathway that plant employs for the production of limonoids remains unexplored for this wonder tree. Results Herein, we report the tracing of limonoid biosynthetic pathway through feeding experiments using C-13 isotopologues of glucose in neem cell suspension. Growth and development specific limonoid spectrum of neem seedling and time dependent limonoid biosynthetic characteristics of cell lines were established. Further to understand the role of mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways in limonoid biosynthesis, Ultra Performance Liquid Chromatography (UPLC)- tandem mass spectrometry based structure-fragment relationship developed for limonoids and their isotopologues have been utilized. Analyses of labeled limonoid extract lead to the identification of signature isoprenoid units involved in azadirachtin and other limonoid biosynthesis, which are found to be formed through mevalonate pathway. This was further confirmed by treatment of cell suspension with mevinolin, a specific inhibitor for MVA pathway, which resulted in drastic decrease in limonoid levels whereas their biosynthesis was unaffected with fosmidomycin mediated plastidial methylerythritol 4-phosphate (MEP) pathway inhibition. This was also conspicuous, as the expression level of genes encoding for the rate-limiting enzyme of MVA pathway, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) was comparatively higher to that of deoxyxylulose-phosphate synthase (DXS) of MEP pathway in different tissues and also in the in vitro grown cells. Thus, this study will give a comprehensive understanding of limonoid biosynthetic pathway with differential contribution of MVA and MEP pathways. Conclusions Limonoid biosynthesis of neem tree and cell lines have been unraveled through comparative quantification of limonoids with that of neem tree and through C-13 limonoid isotopologues analysis. The undifferentiated cell lines of neem suspension produced a spectrum of C-seco limonoids, similar to parental tissue, kernel. Azadirachtin, a C-seco limonoid is produced in young tender leaves of plant whereas in the hard mature leaves of tree, ring intact limonoid nimocinol accumulates in high level. Furthermore, mevalonate pathway exclusively contributes for isoprene units of limonoids as evidenced through stable isotope labeling and no complementation of MEP pathway was observed with mevalonate pathway dysfunction, using chemical inhibitors.1 aAarthy, Thiagarayaselvam1 aMulani, Fayaj, A.1 aPandreka, Avinash1 aKumar, Ashish1 aNandikol, Sharvani, S.1 aHaldar, Saikat1 aThulasiram, Hirekodathakallu, V. uhttps://library.ncl.res.in/content/tracing-biosynthetic-origin-limonoids-and-their-functional-groups-through-stable-isotope02952nas a2200349 4500008004100000022001400041245011300055210006900168260000800237300001100245490000800256520180300264653002202067653003202089653002702121653001402148653001402162653001602176653001802192100002202210700002102232700001802253700002902271700002202300700002602322700002402348700002602372700002102398700002202419700003302441856012802474 2021 eng d a0031-942200aLimonoid biosynthesis 3: functional characterization of crucial genes involved in neem limonoid biosynthesis0 aLimonoid biosynthesis 3 functional characterization of crucial g cAPR a1126690 v1843 a

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-3 beta-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-3 beta-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-3 beta-ol was observed when AiSQE1 was coexpressed 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.

10a24-dien-3 beta-ol10aAzadirachta indica A. Juss.10aCytochrome P450 system10aLimonoids10aMeliaceae10aTirucalla-710aTranscriptome1 aPandreka, Avinash1 aChaya, Patil, S.1 aKumar, Ashish1 aAarthy, Thiagarayaselvam1 aMulani, Fayaj, A.1 aBhagyashree, Date, D.1 aShilpashree, H., B.1 aJennifer, Cheruvathur1 aPonnusamy, Sudha1 aNagegowda, Dinesh1 aThulasiram, Hirekodathakallu uhttps://library.ncl.res.in/content/limonoid-biosynthesis-3-functional-characterization-crucial-genes-involved-neem-limonoid00940nas a2200253 4500008004100000022001400041245013700055210006900192260000800261300001100269490000800280100002200288700002100310700001800331700002900349700002200378700002600400700002400426700002600450700002100476700002200497700003700519856013000556 2021 eng d a0031-942200aLimonoid biosynthesis 3: functional characterization of crucial genes involved in neem limonoid biosynthesis (vol 184, 112669, 2021)0 aLimonoid biosynthesis 3 functional characterization of crucial g cJUL a1127510 v1871 aPandreka, Avinash1 aChaya, Patil, S.1 aKumar, Ashish1 aAarthy, Thiagarayaselvam1 aMulani, Fayaj, A.1 aBhagyashree, Date, D.1 aShilpashree, H., B.1 aJennifer, Cheruvathur1 aPonnusamy, Sudha1 aNagegowda, Dinesh1 aThulasiram, Hirekodathakallu, V. uhttps://library.ncl.res.in/content/limonoid-biosynthesis-3-functional-characterization-crucial-genes-involved-neem-limonoid-002505nas a2200265 4500008004100000022001400041245016800055210006900223260000800292300001200300490000700312520152900319653003101848653001101879653002501890653002201915653002001937100003701957700003801994700002202032700001802054700002002072700002202092856012502114 2023 eng d a1479-262100aTargeted metabolite profiling and de novo transcriptome sequencing reveal the key terpene synthase genes in medicinally important plant, Couroupita guianensis Aubl0 aTargeted metabolite profiling and de novo transcriptome sequenci cDEC a558-5700 v213 a

The Lecythidaceae family tree, Couroupita guianensis Aubl, popularly known as Nagpushpa, is a widely cultivated ornamental tree with several uses in traditional medicine. The tree is revered as highly sacred in Indian traditional culture due to its uniquely shaped, fragrant flowers. Considering the significance, we were prompted to carry out the metabolite and transcriptome analysis of Nagapushpa. The flower, petals, stamen, stem and leaf of C. guianensis were metabolically profiled, and it was discovered that the flower tissue contained the highest terpenoid reservoir. A number of terpenoid pathway transcripts were also found in the flower tissue after transcriptome profiling. KEGG pathway mapping was carried out to correlate transcript sequences with the biosynthesis of different types of terpenes. We were able to clone three full-length terpene synthase gene candidates, i.e. monoterpene ocimene synthase, diterpene ent-kaurene synthase and sesquiterpene farnesene synthase. The transcript expression of selected terpene synthase genes was also verified in flower tissue. These cloned sequences were used for in silico structural investigations and protein function prediction at the level of 3D structure. The data presented in this study provide a comprehensive resource for the metabolic and transcriptomic profiles of C. guianensis. The study paves the way towards the elucidation of terpene biosynthetic pathway in C. guianensis and heterologous production of useful terpenoids in the future.

10aCouroupita guianensis Aubl10aflower10aMetabolite profiling10aterpene synthases10aTranscriptomics1 aThulasiram, Hirekodathakallu, V.1 aKaregaonkar, Shrikant, Jagannathr1 aSharma, Poojadevi1 aKumar, Ashish1 aRamkumar, Sudha1 aPandreka, Avinash uhttps://library.ncl.res.in/content/targeted-metabolite-profiling-and-de-novo-transcriptome-sequencing-reveal-key-terpene