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SummaryEuphorbia grantiiandEuphorbia tirucalliknown to synthesize diverse triterpenes including euphol and tirucallol. These two triterpenes known to possess potent anti-cancer, anti-bacterial, and anti-fungal properties along with various other biological activities. In this study, De-novo assembly and comparative transcriptome analysis of leaf and stem tissues ofE. tirucalliandE. grantiiwere carried out to identify thirteen triterpene synthases from 1,40,227 in correlation with the metabolic profiling. Comparative transcriptome analysis indicated that EutTTS4 and EutTTS5 genes which encodes for euphol/tirucallol and tirucallol synthase were highly expressed in leaf and stem tissue. The genes which encodes α-amyrin synthase (EutTTS1) and lupeol synthase (EutTTS2) were characterized by overexpressing them in YPH499 yeast strain. We have developed using hem1 and erg7 knock yeast strain of lanosterol deficient yeast (TMBL17) and used for over expression of friedelin synthase (EutTTS3), and two novel triterpenes synthases such as euphol/tirucallol synthase (EutTTS4) and tirucallol synthase (EutTTS5). These results are very useful in large scale production of triterpenes by genomic integration of respective triterpene synthases in TMBL yeast strain developed in this study.Significance StatementWe have functionally characterized triterpene synthases fromE. tirucalliandE. grantiiand developed a hem1 and erg7 knock out of lanosterol deficient yeast (TMBL17) for the large-scale production of triterpene and triterpene related products.

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 13C 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 13C 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. Electronic supplementary material The online version of this article (10.1186/s12870-018-1447-6) contains supplementary material, which is available to authorized users.

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.

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 ΙΙ 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. Electronic supplementary material The online version of this article (doi:10.1186/s12870-015-0593-3) contains supplementary material, which is available to authorized users.

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, β-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.

Isolation of Neem triterpenoids from seed kernel and pericarp. Methods 2. Characterization of purified Neem triterpenoids. Figure S1. TLC profile of crude extracts and purified triterpenoids (developed in 70 % ethyl acetate in n-hexane for twice). Figure S2. UPLC-ESI(+)-quadrupole/orbitrap-MS extracted ion chromatograms of the fifteen pure triterpenoids from Neem. Chromatograms have been arranged in the order of increasing retention time. Figure S3. ESI(+)-quadrupole/orbitrap-MS spectra of the fifteen pure triterpenoids from Neem. Figure S4. Standard graphs for the purified triterpenoids prepared in UPLC-ESI(+)-quadrupole/orbitrap-MS; concentration range 0.040-0.003 mg/mL, injection volume 5 μL. Figure S5. Representative UPLC-ESI(+)-quadrupole/orbitrap-MS chromatograms of various Neem tissue extracts (× denotes non-triterpenoids with molecular mass less than 350). Figure S6. Quantitative abundance of individual triterpenoids in different tissues of Neem. Figure S7. Multiple sequence alignment of A. indica geranyl diphosphate synthases (AiGDS). Figure S8. Multiple sequence alignment of A. indica farnesyl diphoshate synthase (AiFDS). Figure S9. Multiple sequence alignment of A. indica Squalene synthase (AiSQS); Amino acid sequence alignment of C. annuum (CaSQS, AAD20626), N. tabacum (NtSQS, AAB08578), A. indica (AiSQS, AFJ15526), L. japonicas (LjSQS, BAC56854), G. max (GmSQS, NP_001236365), P. vulgaris (PvSQS, AHA84150). The solid lines indicate four highly conserved regions 1, 2, 3 and 4 which are considered to be the catalytic sites of squalene synthases. Figure S10. Phylogenetic analysis of AiGDS, AiFDS and AiSQS. Figure S11. Purification of recombinant AiGDS, AiFDS and AiSQS. Table S1. Predicted genes for Triterpenoid back bone biosynthesis. Table S2. Present Identity Matrix of AiGDS with plant homomeric GDS and heteromeric GDS Larger subunits. Table S3. Primers and vectors used for cloning of AiGDS, AiFDS and AiSQS and RT-PCR primers of 18S rRNA, GAPDH, Neem_transcript_10001, AiGDS and AiSQS. Table S4. Buffers used for AiGDS, AiFDS and AiSQS protein purification. Table S5. TargetP analysis Neem_transcript_10912 (AiGDS) and Neem_Transcript_10001. (DOCX 3387 kb)