96 research outputs found
Combinatorial biochemistry of triterpene saponins in plants
No full textPlants are capable of synthesizing an overwhelming variety of secondary metabolites, many of which possess biological activities relevant for the pharmaceutical and chemical industries. Furthermore, there is an ever increasing demand for novel compounds, due to, among others, the growing drug tolerance and resistance in microorganisms and newly emerging diseases. In microorganisms, combinatorial biochemistry is a widely used tool to increase structural variation in several classes of (microbial) natural products. Despite the potential importance of plant secondary metabolites, only a limited fraction of these molecules is currently used, mostly due to their complex structure and the low production levels in planta. Metabolic engineering of plants has offered limited help because the molecular mechanisms steering plant secondary metabolism remain poorly characterized. Here, we used a functional genomics approach to identify candidate genes involved in the saponin biosynthesis of five different plants. After targeted metabolite profiling confirmed the induction of triterpene saponin biosynthesis by methyl jasmonate treatment, a genome-wide cDNA-AFLP transcript profiling was carried out for the five plants. Taking into account the putative functional annotation and the expression pattern of the visualized transcript tags, a set of 259 candidate genes potentially involved in saponin biosynthesis and its regulation were identified. The generated gene list provided the basis for a combinatorial biochemistry platform that targets triterpene saponins in plants. Proof of concept of combinatorial biochemistry was achieved by heterologous expression of the candidate saponin biosynthesis genes in M. truncatula hairy roots. Three of the generated transgenic hairy root lines were found to accumulate novel molecules, two of which were shown to be novel triterpene saponins, whereas the third line produced a set of novel, non-saponin compounds. Furthermore, the identified transcription factors and other regulators were lead candidates for studies investigating the control of the saponin biosynthesis in planta. This led to the identification of a RING membrane-anchor E3 ubiquitin ligase, MAKIBISHI1 (MKB1), that targets 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the enzyme catalyzing the rate-limiting step in the mevalonate pathway, for ubiquitin-mediated proteasomal degradation, thereby controlling saponin biosynthesis
System design and orbit analysis for SpooQySat-1.
No full textThe Center for Quantum Technologies (CQT), which belongs to the National University of Singapore (NUS) is developing a nanosatellite to host their miniaturized Quantum Key Distribution (QKD) payload called SPEQS. QKD makes it possible to generate encryption keys through the use of strongly correlated photons. This would allow two parties to securely communicate with each other without any possibility of their conversation being hacked. CQT has already demonstrated a QKD free space-link in 2005. The main challenge now lies within the creation of a global QKD network. One possible way to do this is through the use of nanosatellites. As CQT has no experience in designing nanosatellites, a first step towards this goal is to design a satellite which allows the team to test a version of their payload. The main purpose of the thesis work is to present a reliable design for CQT’s first nanosatellite, SpooQySat-1, through the implementation of a systems engineering approach. The requirements as first defined by CQT are revised and redefined through the use of a requirements discovery tree. After the requirements list was complete, an orbit analysis has been carried out to ensure the best suitable orbit was selected for the mission. This, together with the requirements, served as input for the design analysis. A concept of operations has been generated, leading to a realistic power budget that has been compared to the estimated incoming power for various cases. A communication analysis has been carried out taking into account the noisy environment of Singapore. COTS component selection has been carried out for all subsystems. To ensure maximum reliability, a risk analysis has been implemented. Data regarding CubeSat failures has been gathered to inform the risk analysis. The outcome of the risk analysis was a mitigation strategy that allowed a new design iteration to ensure maximum reliability.Space System EngineeringAerospace Engineerin
Metabolite profiling of plant tissues by liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry
No full textPlants accumulate an overwhelming variety of secondary metabolites that play important roles in defense and interaction of the plant with its environment. To investigate the dynamics of plant secondary metabolism, large-scale untargeted metabolite profiling (metabolomics) is mandatory. Here, we describe a detailed protocol for untargeted metabolite profiling in which methanol extracts of jasmonate-treated plant tissues are analyzed by reversed-phase liquid chromatography coupled to negative-ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (MS). By means of dedicated integration and alignment software, the relative abundance of thousands of mass peaks, corresponding to hundreds of compounds, is calculated, and mass peaks of which the area is significantly changed by jasmonate treatment are identified. Subsequently, the metabolites corresponding to the significantly changed peaks are tentatively annotated using the accurate mass prediction of the Fourier transform-MS and the generated MS/MS data. Via this method, compounds of medium polarity, such as glucosinolates, alkaloids, phenylpropanoids, flavonoids, polyamines, and saponins, can be analyzed
Analysis of RNA-Seq data with TopHat and Cufflinks for genome-wide expression analysis of jasmonate-treated plants and plant cell cultures
No full textThe recent development of various deep sequencing techniques has led to the most powerful transcript profiling method available to date, RNA sequencing or RNA-Seq. Besides the identification of new genes and new splice variants of known genes, RNA-Seq allows to compare the whole transcriptome of any organism under two or more experimental conditions, such as before and after jasmonate treatment. However, the vast amounts of data generated during RNA-Seq experiments require complex computational methods for read mapping and expression quantification. Here, we describe a detailed protocol for the analysis of deep sequencing data, starting from the raw RNA-Seq reads. First, a quality check is performed on the raw reads to assess the quality of the sequencing. Subsequently, adapters and low-quality sequences are trimmed off the raw reads. The resulting processed reads are mapped to the reference genome, and the mapped reads are counted to generate expression data for the annotated genes for each sample. This method can be used for the analysis of RNA-Seq data of any organism for which a reference genome is available
Metabolite profiling of Triterpene Saponins in medicago truncatula hairy roots by liquid chromatography fourier transform Ion Cyclotron resonance mass spectrometry
No full textTriterpenes are one of the largest classes of plant natural products, with an enormous variety in structure and bioactivities. Here, triterpene saponins from hairy roots of the model legume Medicago truncatula were profiled with reversed-phase liquid chromatography coupled to negative-ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (LC ESI FT-ICR MS). Owing to the accuracy of the FT-ICR MS, reliable molecular formulas of the detected compounds could be predicted, which, together with the generated MS" spectra, allowed the tentative identification of 79 different saponins, of which 61 had not been detected previously in M. truncatula. Upon collision-induced dissociation of saponins that contain a uronic acid residue in the sugar chain, fragment ions resulting from cross-ring cleavages of the uronic acid residues were observed. The identified saponins are glycosides of 10 different sapogenins, of which three were not detected before in M. truncatula. Zanhic acid glycosides, which are prevalent in the aerial parts of M. truncatula, were absent in the hairy root extract's. This metabolite compendium will facilitate future functional genomic studies of triterpene saponin biosynthesis in M. truncptula
ChemInform Abstract: Combinatorial Biosynthesis in Plants: A (P)review on Its Potential and Future Exploitation
No full textCombinatorial biosynthesis in plants: a (p)review on its potential and future exploitation
No full textCombinatorial biochemistry, also called combinatorial biosynthesis, comprises a series of methods that establish novel enzyme-substrate combinations in vivo and, in turn, lead to the biosynthesis of new, natural product-derived compounds that can be used in drug discovery programs. Plants are an extremely rich source of bioactive natural products and continue to possess a huge potential for drug discovery. In this review, we discuss the state-of-the-art in combinatorial biosynthesis methods to generate novel molecules from plants. We debate on the progress and potential in biotransformation, mutasynthesis, combinatorial metabolism in hybrids, activation of silent plant metabolism and synthetic biology in plants to create opportunities for the combinatorial biosynthesis of plant-derived natural products, and, ultimately, for drug discovery. The therapeutic value of two classes of natural products, the terpenoid indole alkaloids and the triterpene saponins, is particularly highlighted
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