@article { ISI:000305379000012, title = {Transformation of (+/-)-lavandulol and (+/-)-tetrahydrolavandulol by a fungal strain Rhizopus oryzae}, journal = {Bioresource Technology}, volume = {115}, year = {2012}, month = {JUL}, pages = {70-74}, publisher = {ELSEVIER SCI LTD}, address = {THE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, OXON, ENGLAND}, abstract = {

Biotransformation of an irregular monoterpene alcohol, (+/-)-lavandulol [(+/-)-5-methyl-2-(1-methylethenyl)-4-hexen-1-ol] (I) and its tetrahydro derivative, (+/-)-tetrahydrolavandulol [(+/-)-2-isopropyl-5-methylhexan-1-ol] (II) were studied using a soil isolated fungal strain Rhizopus oryzae. Five metabolites, 2-((3,3-dimethyloxiran-2-yl)methyl)-3-methylbut-3-en-1-ol (Ia), 2-methyl-5-(prop-1-en-2-yl)hex-2ene-1,6-diol (Ib), 2-methyl-5-(prop-1-en-2-yl)hexane-1,6-diol (Ic), 2-(3-methylbut-2-enyl)-3-methylenebutane-1,4-diol (Id), 5-methyl-2-(2-methyloxiran-2-yl)hex-4-en-1-01 (Ie) have been isolated from the fermentation medium and characterized with lavandulol as a substrate. When tetrahydrolavandulol used as a substrate, two metabolites 2-isopropyl-5-methylhexane-1,5-diol (IIa) and 2-isopentyl-3-methylbutane-1,3-diol (lib) have been isolated from the fermentation medium. Biotransformation studies with R. oryzae clearly indicate that the organism initiates the transformation either by hydroxylation at allylic methyl groups or epoxidation of double bond. GC and GCMS analyses indicated that both (R)and (S)-enantiomers of I and II have been transformed into corresponding hydroxylated or epoxy derivatives, when racemic I and II were used as substrates. (C) 2011 Elsevier Ltd. All rights reserved.

}, keywords = {Biotransformation, Fungus, Lavandulol, Rhizopus oryzae, Tetrahydrolavandulol}, issn = {0960-8524}, doi = {10.1016/j.biortech.2011.11.038}, author = {Daramwar, Pankaj P. and Rincy, Raju and Niloferjahan, Siddiqui and Krithika, Ramakrishnan and Gulati, Arvind and Yadav, Amit and Sharma, Rakesh and Thulasiram, Hirekodathakallu V.} } @article { ISI:000360677400002, title = {Triterpenoid profiling and functional characterization of the initial genes involved in isoprenoid biosynthesis in neem (Azadirachta indica)}, journal = {BMC Plant Biology}, volume = {15}, year = {2015}, month = {SEP}, pages = {Article Number: 214}, publisher = {BIOMED CENTRAL LTD}, address = {236 GRAYS INN RD, FLOOR 6, LONDON WC1X 8HL, ENGLAND}, abstract = {

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.

}, keywords = {Azadirachta indica, Quantitative profiling, Transcriptome, Triterpenoids}, issn = {1471-2229}, doi = {10.1186/s12870-015-0593-3}, author = {Pandreka, Avinash and Dandekar, Devdutta S. and Haldar, Saikat and Uttara, Vairagkar and Vijayshree, Shinde G. and Mulani, Fayaj A. and Aarthy, Thiagarayaselvam and Thulasiram, Hirekodathakallu V.} } @article {45420, title = { Tracing the biosynthetic origin of limonoids and their functional groups through stable isotope labeling and inhibition in neem tree (Azadirachta indica) cell suspension}, journal = {BMC Plant Biology}, volume = {18}, year = {2018}, month = {OCT}, type = {Article}, abstract = {Background 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.}, isbn = {1471-2229}, doi = {10.1186/s12870-018-1447-6}, author = {Aarthy, Thiagarayaselvam and Mulani, Fayaj A. and Pandreka, Avinash and Kumar, Ashish and Nandikol, Sharvani S. and Haldar, Saikat and Thulasiram, Hirekodathakallu V.} } @article {45524, title = {Terpene profiling, transcriptome analysis and characterization of cis{\textendash}terpineol synthase from Ocimum}, journal = {Physiology and Molecular Biology of Plants}, volume = {25}, year = {2019}, month = {JAN}, pages = {47-57}, type = {Article}, abstract = {

Ocimum species produces a varied mix of different metabolites that imparts immense medicinal properties. To explore this chemo-diversity, we initially carried out metabolite profiling of different tissues of five Ocimum species and identified the major terpenes. This analysis broadly classified these five Ocimum species into two distinct chemotypes namely, phenylpropanoid-rich and terpene-rich. In particular, -caryophyllene, myrcene, limonene, camphor, borneol and selinene were major terpenes present in these Ocimum species. Subsequently, transcriptomic analysis of pooled RNA samples from different tissues of Ocimum gratissimum, O. tenuiflorum and O. kilimandscharicum identified 38 unique transcripts of terpene synthase (TPS) gene family. Full-length gene cloning, followed by sequencing and phylogenetic analysis of three TPS transcripts were carried out along with their expression in various tissues. Terpenoid metabolite and expression profiling of candidate TPS genes in various tissues of Ocimum species revealed spatial variances. Further, putative TPS contig 19414 (TPS1) was selected to corroborate its role in terpene biosynthesis. Agrobacterium-mediated transient over-expression assay of TPS1 in the leaves of O. kilimandscharicum and subsequent metabolic and gene expression analyses indicated it as a cis{\textendash}terpineol synthase. Overall, present study provided deeper understanding of terpene diversity in Ocimum species and might help in the enhancement of their terpene content through advanced biotechnological approaches.

}, keywords = {Agro-infiltration assay, Metabolite profiling, Ocimum, Terpene synthase, Terpenes, Transcriptome}, issn = {0971-5894}, doi = {10.1007/s12298-018-0612-6}, author = {Anand, Atul and Jayaramaiah, Ramesha H. and Beedkar, Supriya D. and Dholakia, Bhushan B. and Lavhale, Santosh G. and Punekar, Sachin A. and Gade, Wasudeo N. and Thulasiram, Hirekodathakallu V. and Giri, Ashok P.} } @article {49083, title = {Targeted metabolite profiling and de novo transcriptome sequencing reveal the key terpene synthase genes in medicinally important plant, Couroupita guianensis Aubl}, journal = {Plant Genetic Resources-Characterization and Utilization}, volume = {21}, year = {2023}, month = {DEC}, pages = {558-570}, type = {Article}, abstract = {

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.

}, keywords = {Couroupita guianensis Aubl, flower, Metabolite profiling, terpene synthases, Transcriptomics}, issn = {1479-2621}, doi = {10.1017/S1479262123000953}, author = {Thulasiram, Hirekodathakallu V. and Karegaonkar, Shrikant Jagannathrao and Sharma, Poojadevi and Kumar, Ashish and Ramkumar, Sudha and Pandreka, Avinash} }