Development of CE-C4D Method for Determination Tropane Alkaloids.
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ABSTRACT:
SUBMITTER: Golab M
PROVIDER: S-EPMC8510007 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Tropane alkaloids from nightshade plants are neurotransmitter inhibitors that are used for treating neuromuscular disorders and are classified as essential medicines by the World Health Organization1,2. Challenges in global supplies have resulted in frequent shortages of these drugs3,4. Further vulnerabilities in supply chains have been revealed by events such as the Australian wildfires5 and the COVID-19 pandemic6. Rapidly deployable production strategies that are robust to environmental and socioeconomic upheaval7,8 are needed. Here we engineered baker's yeast to produce the medicinal alkaloids hyoscyamine and scopolamine, starting from simple sugars and amino acids. We combined functional genomics to identify a missing pathway enzyme, protein engineering to enable the functional expression of an acyltransferase via trafficking to the vacuole, heterologous transporters to facilitate intracellular routing, and strain optimization to improve titres. Our integrated system positions more than twenty proteins adapted from yeast, bacteria, plants and animals across six sub-cellular locations to recapitulate the spatial organization of tropane alkaloid biosynthesis in plants. Microbial biosynthesis platforms can facilitate the discovery of tropane alkaloid derivatives as new therapeutic agents for neurological disease and, once scaled, enable robust and agile supply of these essential medicines.
| S-EPMC7529995 | biostudies-literature
Project description:Mycotoxins and pesticides regularly co-occur in agricultural products worldwide. Thus, humans can be exposed to both toxic contaminants and pesticides simultaneously, and multi-methods assessing the occurrence of various food contaminants and residues in a single method are necessary. A two-dimensional high performance liquid chromatography tandem mass spectrometry method for the analysis of 40 (modified) mycotoxins, two plant growth regulators, two tropane alkaloids, and 334 pesticides in cereals was developed. After an acetonitrile/water/formic acid (79:20:1, v/v/v) multi-analyte extraction procedure, extracts were injected into the two-dimensional setup, and an online clean-up was performed. The method was validated according to Commission Decision (EC) no. 657/2002 and document N° SANTE/12682/2019. Good linearity (R2 > 0.96), recovery data between 70-120%, repeatability and reproducibility values < 20%, and expanded measurement uncertainties < 50% were obtained for a wide range of analytes, including very polar substances like deoxynivalenol-3-glucoside and methamidophos. However, results for fumonisins, zearalenone-14,16-disulfate, acid-labile pesticides, and carbamates were unsatisfying. Limits of quantification meeting maximum (residue) limits were achieved for most analytes. Matrix effects varied highly (-85 to +1574%) and were mainly observed for analytes eluting in the first dimension and early-eluting analytes in the second dimension. The application of the method demonstrated the co-occurrence of different types of cereals with 28 toxins and pesticides. Overall, 86% of the samples showed positive findings with at least one mycotoxin, plant growth regulator, or pesticide.
| S-EPMC8044062 | biostudies-literature
Project description:We analyzed the transcriptome of A. acutangulus roots by deep RNA sequencing to dig TAs biosynthetic genes. KOG (Eukaryotic Orthologous Groups) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses identified 48 unigenes referring to the tropane, piperidine and pyridine alkaloid biosynthesis, 145 unigenes presumably involved in distribution of arginine to TAs biosynthesis, and 86 unigenes referring to the terpenoid backbone biosynthesis. Furthermore, 82 unigenes annotated as cytochrome P450 family members seemed to be involved in secondary metabolism pathways. Previously unknown TAs biosynthetic genes in A. acutangulus, which encode littorine mutase/monooxygenase (CYP80F1) and diamine oxidase (DAO), were identified by this study.
2016-07-08 | GSE62162 | GEO
Project description:BackgroundPlants have evolved physical-chemical defense to prevent/diminish damage by their enemies. Chemical defense involves the synthesis' pathways of specialized toxic, repellent, or anti-nutritive metabolites to herbivores. Molecular evolutionary studies have revealed the origin of new genes, acquisition and functional diversification along time in different plant lineages.MethodsUsing bioinformatic tools we analyze gene divergence of tropane alkaloids (TAs) and terpene synthases (TPSs) in Datura stramonium and other species of Solanaceae; compared gene and amino acids sequence of TAs and TPSs on genomes, cDNA and proteins sequences of Viridiplantae. We analyzed two recently assembled genomes of D. stramonium (Ticumán and Teotihuacán), transcriptomes of Datura metel and genomes of other Solanaceae. Hence, we analyzed variation of TAs and TPSs to infer genes involved in plant defense and plant responses before stress. We analyzed protein modeling and molecular docking to predict interactions between H6H and ligand; we translated the sequences (Teo19488, Tic8550 and Tic8549) obtained from the two genomes of D. stramonium by using Swiss-Model and Ramachandran plot and MolProbity structure validation of Teo19488 protein model.ResultsFor TAs, we detected an expansion event in the tropinone reductase II (TRII) and the ratio synonymous/non-synonymous substitutions indicate positive selection. In contrast, a contraction event and negative selection was detected in tropinone reductase I (TRI). In Hy-oscyamine 6 b-hydroxylase (H6H), enzyme involved in the production of tropane alkaloids atropine and scopolamine, the synonymous/non-synonymous substitution ratio in its dominion indicates positive selection. For terpenes (TPS), we found 18 DsTPS in D. stramomiun and seven in D. metel; evolutionary analyses detected positive selection in TPS10.1 and TPS10.2 of D. stramonium and D. metel. Comparison of copies of TPSs in D. stramonium detected variation among them in the binding site. Duplication events and differentiation of TAs and TPSs of D. stramonium, as compared to other Solanaceae, suggest their possible involvement on adaptive evolution of defense to herbivores. Protein modeling and docking show that the three protein structures obtained of DsH6H from Teo19488, Tic-8550 and Tic8549 maintain the same interactions and the union site of 2OG-FeII_Oxy with the Hy-o ligand as in 6TTM of D. metel.ConclusionOur results indicate differences in the number of gene copies involved in the synthesis of tropane alkaloids, between the genomes of D. stramonium from two Mexican populations. More copies of genes related to the synthesis of tropane alkaloids (TRI, TRII, H6H, PMT) are found in D. stramonium as compared to Viridiplantae. Likewise, for terpene synthases (TPS), TPS-10 is duplicated in D. stramonium and D. metel. Further studies should be directed to experimentally assess gain (overexpression) or loss (silencing) of function of duplicated genes.
| S-EPMC8212831 | biostudies-literature
Project description:Tropane alkaloids (TAs) are a class of phytochemicals produced by plants of the nightshade family used for treating diverse neurological disorders. Here, we demonstrate de novo production of tropine, a key intermediate in the biosynthetic pathway of medicinal TAs such as scopolamine, from simple carbon and nitrogen sources in yeast (Saccharomyces cerevisiae). Our engineered strain incorporates 15 additional genes, including 11 derived from diverse plants and bacteria, and 7 disruptions to yeast regulatory or biosynthetic proteins to produce tropine at titers of 6 mg/L. We also demonstrate the utility of our engineered yeast platform for the discovery of TA derivatives by combining biosynthetic modules from distant plant lineages to achieve de novo production of cinnamoyltropine, a non-canonical TA. Our engineered strain constitutes a starting point for future optimization efforts towards realizing industrial fermentation of medicinal TAs and a platform for the synthesis of TA derivatives with enhanced bioactivities.
| S-EPMC6690885 | biostudies-other
Project description:Theories of plant invasion based on enemy release in a new range assume that selection exerted by specialist herbivores on defence traits should be reduced, absent, or even selected against in the new environment. Here, we measured phenotypic selection on atropine and scopolamine concentration of Datura stramonium in eight native (Mexico) and 14 non-native (Spain) populations. Native populations produced between 20 and 40 times more alkaloid than non-native populations (atropine: 2.0171 vs. 0.0458 mg/g; scopolamine: 1.004 vs. 0.0488 mg/g, respectively). Selection on alkaloids was negative for atropine and positive for scopolamine concentration in both ranges. However, the effect sizes of selection gradients were only significant in the native range. Our results support the assumption that the reduction of plant defence in the absence of the plant's natural enemies in invasive ranges is driven by natural selection.
| S-EPMC6787939 | biostudies-literature
Project description:The skeleton of tropane alkaloids is derived from ornithine-derived N-methylpyrrolinium and two malonyl-CoA units. The enzymatic mechanism that connects N-methylpyrrolinium and malonyl-CoA units remains unknown. Here, we report the characterization of three pyrrolidine ketide synthases (PYKS), AaPYKS, DsPYKS, and AbPYKS, from three different hyoscyamine- and scopolamine-producing plants. By examining the crystal structure and biochemical activity of AaPYKS, we show that the reaction mechanism involves PYKS-mediated malonyl-CoA condensation to generate a 3-oxo-glutaric acid intermediate that can undergo non-enzymatic Mannich-like condensation with N-methylpyrrolinium to yield the racemic 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid. This study therefore provides a long sought-after biosynthetic mechanism to explain condensation between N-methylpyrrolinium and acetate units and, more importantly, identifies an unusual plant type III polyketide synthase that can only catalyze one round of malonyl-CoA condensation.
| S-EPMC6731253 | biostudies-literature
Project description:Plant alkaloids constitute an important class of bioactive chemicals with applications in medicine and agriculture. However, the knowledge gap of the diversity and biosynthesis of phytoalkaloids prevents systematic advances in biotechnology for engineered production of these high-value compounds. In particular, the identification of cytochrome P450s driving the structural diversity of phytoalkaloids has remained challenging. Here, we use a combination of reverse genetics with discovery metabolomics and multivariate statistical analysis followed by in planta transient assays to investigate alkaloid diversity and functionally characterize two candidate cytochrome P450s genes from Atropa belladonna without a priori knowledge of their functions or information regarding the identities of key pathway intermediates. This approach uncovered a largely unexplored root localized alkaloid sub-network that relies on pseudotropine as precursor. The two cytochrome P450s catalyze N-demethylation and ring-hydroxylation reactions within the early steps in the biosynthesis of diverse N-demethylated modified tropane alkaloids.
| S-EPMC9250511 | biostudies-literature
Project description:Under hypoxic conditions, the expression of Vitreoscilla hemoglobin (VHb) in plants is proposed to increase the productivity of certain oxygen-requiring metabolic pathways by promoting the delivery of oxygen. Tropane alkaloids (TAs) are a class of important plant secondary metabolites with significant medicinal value; the final step in their biosynthesis requires oxygen. Whether heterologous expression of VHb, especially in different subcellular compartments, can accelerate the accumulation of TAs is not known. Herein, the effect of heterologous expression of VHb in different subcellular locations on the TA profile of H. niger hairy roots was investigated. The targeted expression of VHb in the plastids (using pVHb-RecA construct), led to the accumulation of 197.68??g/g hyoscyamine in the transgenic H. niger hairy roots, which was 1.25-fold of the content present in the lines in which VHb expression was not targeted, and 3.66-fold of that present in the wild type (WT) lines. The content of scopolamine was increased by 2.20- and 4.70-fold in the pVHb-RecA transgenic lines compared to that in the VHb transgenic and WT lines. Our results demonstrate that VHb could stimulate the accumulation of TAs in the transgenic H. niger hairy roots. Quantitative RT-PCR analysis revealed that the expression of key genes involved in TA biosynthesis increased significantly in the VHb transgenic lines. We present the first description of a highly efficient strategy to increase TA content in H. niger. Moreover, our results also shed light on how the production of desired metabolites can be efficiently enhanced by using more accurate and appropriate genetic engineering strategies.
| S-EPMC6299274 | biostudies-literature
Project description:It has been recognized that other than habitat loss, degradation and fragmentation, the infection of the roundworm Baylisascaris schroederi (B. schroederi) is one of the major causes of death in wild giant pandas. However, the prevalence and intensity of the parasite infection has been inconsistently reported through a method that uses sedimentation-floatation followed by a microscope examination. This method fails to accurately determine infection because there are many bamboo residues and/or few B. schroederi eggs in the examined fecal samples. In the present study, we adopted a method that uses PCR and capillary electrophoresis combined with a single-strand conformation polymorphism analysis (PCR/CE-SSCP) to detect B. schroederi infection in wild giant pandas at a nature reserve, and compared it to the traditional microscope approach. The PCR specifically amplified a single band of 279-bp from both fecal samples and positive controls, which was confirmed by sequence analysis to correspond to the mitochondrial COII gene of B. schroederi. Moreover, it was demonstrated that the amount of genomic DNA was linearly correlated with the peak area of the CE-SSCP analysis. Thus, our adopted method can reliably detect the infectious prevalence and intensity of B. schroederi in wild giant pandas. The prevalence of B. schroederi was found to be 54% in the 91 fecal samples examined, and 48% in the fecal samples of 31 identified individual giant pandas. Infectious intensities of the 91 fecal samples were detected to range from 2.8 to 959.2 units/gram, and from 4.8 to 959.2 units/gram in the fecal samples of the 31 identified giant pandas. For comparison, by using the traditional microscope method, the prevalence of B. schroederi was found to be only 33% in the 91 fecal samples, 32% in the fecal samples of the 31 identified giant pandas, and no reliable infectious intensity was observed.
| S-EPMC3416865 | biostudies-literature