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Blueberries (Vaccinium spp.) are well known for their nutritional quality, and recent work has shown that Vaccinium spp. also produce iridoids, which are specialized metabolites with potent health‐promoting benefits. The iridoid glycoside monotropein, which has anti‐inflammatory and antinociceptive activities, has been detected in several wild blueberry species but in only a few cultivated highbush blueberry cultivars. How monotropein is produced in blueberry and the genes involved in its biosynthesis remain to be elucidated. Using a monotropein‐positive (M+) and monotropein‐negative (M−) cultivar of blueberry, we employed transcriptomics and comparative genomics to identify candidate genes in the blueberry iridoid biosynthetic pathway. Orthology analysis was completed using de novo transcript assemblies for both the M+ and M− blueberry cultivars along with the known iridoid‐producing plant species Catharanthus roseus to identify putative genes involved in key steps in the early iridoid biosynthetic pathway. From the identified orthologs, we functionally characterized iridoid synthase (ISY), a key enzyme involved in formation of the iridoid scaffold, from both the M+ and M− cultivars. Detection of nepetalactol suggests that ISY from both the M+ and M− cultivars produce functional enzymes that catalyze the formation of iridoids. Transcript accumulation of the putative ISY gene did not correlate with monotropein production, suggesting other genes in the monotropein biosynthetic pathway may be more directly responsible for differential accumulation of the metabolite in blueberry. Mutual rank analysis revealed that ISY is co‐expressed with UDP‐glucuronosyltransferase, which encodes an enzyme downstream of the ISY step. Results from this study contribute new knowledge in our understanding of iridoid biosynthesis in blueberry and could lead to development of new cultivars with increased human health benefits. [ABSTRACT FROM AUTHOR]

Dolichol is a lipid that is involved in protein glycosylation, a process that is essential for all eukaryotic life. In this issue of Cell , Wilson and coworkers 1 report how a rare human genetic disorder led to the discovery of dolichol biosynthesis. Dolichol is a lipid that is involved in protein glycosylation, a process that is essential for all eukaryotic life. In this issue of Cell , Wilson and coworkers report how a rare human genetic disorder led to the discovery of dolichol biosynthesis. [ABSTRACT FROM AUTHOR]

The iridoids comprise a large family of distinctive bicyclic monoterpenes that possess a wide range of pharmacological activities, including anticancer, anti-inflammatory, antifungal and antibacterial activities (1-4). Additionally, certain iridoids are used as sex pheromones in agriculturally important species of aphids, a fact that has underpinned innovative and integrated pest management strategies (5). To harness the biotechnological potential of this natural product class, the enzymes involved in the biosynthetic pathway must be elucidated. Here we report the discovery of iridoid synthase, a plant-derived enzyme that generates the iridoid ring scaffold, as evidenced by biochemical assays, gene silencing, co-expression analysis and localization studies. In contrast to all known monoterpene cyclases, which use geranyl diphosphate as substrate and invoke a cationic intermediate, iridoid synthase uses the linear monoterpene 10-oxogeranial as substrate and probably couples an initial NAD(P)H-dependent reduction step with a subsequent cyclization step via a Diels-Alder cycloaddition or a Michael addition. Our results illustrate how a short-chain reductase was recruited as cyclase for the production of iridoids in medicinal plants. Furthermore, we highlight the prospects of using unrelated reductases to generate artificial cyclic scaffolds. Beyond the recognition of an alternative biochemical mechanism for the biosynthesis of cyclic terpenes, we anticipate that our work will enable the large-scale heterologous production of iridoids in plants and microorganisms for agricultural (5-8) and pharmaceutical (1-4,9) applications., The crucial step in the biosynthesis of all cyclic terpenes is the cyclization step that gives rise to the individual ring systems. In all kingdoms of life, this step has [...]

Halogenation, which was once considered a rare occurrence in nature, has now been observed in many natural product biosynthetic pathways (1). However, only a small fraction of halogenated compounds have been isolated from terrestrial plants (2). Given the impact that halogenation can have on the biological activity of natural products (1), we reasoned that the introduction of halides into medicinal plant metabolism would provide the opportunity to rationally bioengineer a broad variety of novel plant products with altered, and perhaps improved, pharmacological properties. Here we report that chlorination biosynthetic machinery from soil bacteria can be successfully introduced into the medicinal plant Catharanthus roseus (Madagascar periwinkle). These prokaryotic halogenases function within the context of the plant cell to generate chlorinated tryptophan, which is then shuttled into monoterpene indole alkaloid metabolism to yield chlorinated alkaloids. A new functional group--a halide--is thereby introduced into the complex metabolism of C. roseus, and is incorporated in a predictable and regioselective manner onto the plant alkaloid products. Medicinal plants, despite their genetic and developmental complexity, therefore seem to be a viable platform for synthetic biology efforts., Numerous halogenase enzymes from soil bacteria have been identified and characterized extensively (1-3-5). Two of these flavoenzymes, PyrH (6,7) and RebH (8-11), chlorinate the indole ring of tryptophan in the [...]

Author(s): Frédéric H. Vaillancourt [1, 3]; Ellen Yeh [1, 3]; David A. Vosburg [1, 2]; Sarah E. O'Connor [1, 2]; Christopher T. Walsh (corresponding author) [1] Enzymatic incorporation of chlorine, [...]

Several pathovars of Pseudomonas syringae produce the phytotoxin coronatine (COR), which contains an unusual amino acid, the 1-amino-2-ethylcyclopropane carboxylic acid called coronamic acid (CMA), which is covalently linked to a polyketide-derived carboxylic acid, coronafacic acid, by an amide bond. The region of the COR biosynthetic gene cluster proposed to be responsible for CMA biosynthesis was resequenced, and errors in previously deposited cmaA sequences were corrected. These efforts allowed overproduction of P. syringae pv. glycinea PG4180 CmaA in P. syringae pv. syringae FF5 as a FLAG-tagged protein and overproduction of P. syringae pv. tomato CmaA in Escherichia coli as a His-tagged protein; both proteins were in an enzymatically active form. Sequence analysis of CmaA indicated that there were two domains, an adenylation domain (A domain) and a thiolation domain (T domain). AT[P-sup.32]P[P.sub.i] exchange assays showed that the A domain of CmaA catalyzes the conversion of branched-chain L-amino acids and ATP into the corresponding aminoacyl-AMP derivatives, with a kinetic preference for L-allo-isoleucine. Additional experiments demonstrated that the T domain of CmaA, which is posttranslationally modified with a 4'-phosphopantetheinyl group, reacts with the AMP derivative of L-allo-isoleucine to produce an aminoacyl thiolester intermediate. This covalent species was detected by incubating CmaA with ATP and L-[G-[sup.3]H]allo.isoleucine, followed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis. It is postulated that the L-allo-isoleucine covalently tethered to CmaA serves as the substrate for additional enzymes in the CMA biosynthetic pathway that catalyze cyclopropane ring formation, which is followed by thiolester hydrolysis, yielding free CMA. The availability of catalytically active CmaA should facilitate elucidation of the details of the subsequent steps in the formation of this novel cyclopropyl amino acid.

Countless reports describe the isolation and structural characterization of natural products, yet this information remains disconnected and underutilized. Using a cheminformatics approach, we leverage the reported observations of iridoid glucosides with the known phylogeny of a large iridoid producing plant family (Lamiaceae) to generate a set of biosynthetic pathways that best explain the extant iridoid chemical diversity. We developed a pathway reconstruction algorithm that connects iridoid reports via reactions and prunes this solution space by considering phylogenetic relationships between genera. We formulate a model that emulates the evolution of iridoid glucosides to create a synthetic data set, used to select the parameters that would best reconstruct the pathways, and apply them to the iridoid data set to generate pathway hypotheses. These computationally generated pathways were then used as the basis by which to select and screen biosynthetic enzyme candidates. Our model was successfully applied to discover a cytochrome P450 enzyme from Callicarpa americana that catalyzes the oxidation of bartsioside to aucubin, predicted by our model despite neither molecule having been observed in the genus. We also demonstrate aucubin synthase activity in orthologues of Vitex agnus-castus , and the outgroup Paulownia tomentosa , further strengthening the hypothesis, enabled by our model, that the reaction was present in the ancestral biosynthetic pathway. This is the first systematic hypothesis on the epi -iridoid glucosides biosynthesis in 25 years and sets the stage for streamlined work on the iridoid pathway. This work highlights how curation and computational analysis of widely available structural data can facilitate hypothesis-based gene discovery. [ABSTRACT FROM AUTHOR]

Research has been conducted on the epothilones, hybrid polyketide/nonribosomal peptide antitumor agents. The expression and purification of Escherichia coli enzymes involved in the epothilone production have been carried out and the results are reported.

Research has been conducted on the amino acids found in proteins and forming peptide antibiotics by specific oxidation at the beta carbon. The strategy in the peptide antibiotic biosynthesis has been developed and discussed.

Many plant species from the Apocynaceae, Loganiaceae and Rubiaceae families evolved a specialized metabolism leading to the synthesis of a broad palette of monoterpene indole alkaloids (MIAs). These compounds are believed to constitute a cornerstone of the plant chemical arsenal but above all several MIAs display pharmacological properties that have been exploited for decades by humans to treat various diseases. It is established that MIAs are produced in planta due to complex biosynthetic pathways engaging a multitude of specialized enzymes but also a complex tissue and subcellular organization. In this context, N -methyltransferases (NMTs) represent an important family of enzymes indispensable for MIA biosynthesis but their characterization has always remained challenging. In particular, little is known about the subcellular localization of NMTs in MIA-producing plants. Here, we performed an extensive analysis on the subcellular localization of NMTs from four distinct medicinal plants but also experimentally validated that two putative NMTs from Catharanthus roseus exhibit NMT activity. Apart from providing unprecedented data regarding the targeting of these enzymes in planta , our results point out an additional layer of complexity to the subcellular organization of the MIA biosynthetic pathway by introducing tonoplast and peroxisome as new actors of the final steps of MIA biosynthesis. [ABSTRACT FROM AUTHOR]