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Engineering of biosynthetic enzymes is increasingly employed to synthesize structural analogues of antibiotics. Of special interest are non-ribosomal peptide synthetases (NRPSs) responsible for production of important antimicrobial peptides. Here, directed evolution of an adenylation domain of a Pro-specific NRPS module completely switched substrate specificity to the non-standard amino acid piperazic acid (Piz) bearing a labile N-N bond. This success was achieved by LC-MS/MS based screening of small, rationally designed mutant libraries and can presumably be replicated with a larger number of substrates and NRPS modules. The evolved NRPS produces a Piz-derived gramicidin S analog. Thus, we give new impetus to the too-early dismissed idea that widely accessible low-throughput methods can switch the specificity of NRPSs in a biosynthetically useful fashion.

Strychnine is a natural product that, through isolation, structural elucidation and synthetic efforts, shaped the field of organic chemistry. Currently, strychnine is used as a pesticide to control rodents1 because of its potent neurotoxicity2,3. The polycyclic architecture of strychnine has inspired chemists to develop new synthetic transformations and strategies to access this molecular scaffold4, yet it is still unknown how plants create this complex structure. Here we report the biosynthetic pathway of strychnine, along with the related molecules brucine and diaboline. Moreover, we successfully recapitulate strychnine, brucine and diaboline biosynthesis in Nicotiana benthamiana from an upstream intermediate, thus demonstrating that this complex, pharmacologically active class of compounds can now be harnessed through metabolic engineering approaches.

Advances in omics technologies now permit the generation of highly contiguous genome assemblies, detection of transcripts and metabolites at the level of single cells and high-resolution determination of gene regulatory features. Here, using a complementary, multi-omics approach, we interrogated the monoterpene indole alkaloid (MIA) biosynthetic pathway in Catharanthus roseus, a source of leading anticancer drugs. We identified clusters of genes involved in MIA biosynthesis on the eight C. roseus chromosomes and extensive gene duplication of MIA pathway genes. Clustering was not limited to the linear genome, and through chromatin interaction data, MIA pathway genes were present within the same topologically associated domain, permitting the identification of a secologanin transporter. Single-cell RNA-sequencing revealed sequential cell-type-specific partitioning of the leaf MIA biosynthetic pathway that, when coupled with a single-cell metabolomics approach, permitted the identification of a reductase that yields the bis-indole alkaloid anhydrovinblastine. We also revealed cell-type-specific expression in the root MIA pathway.

Vinblastine is a chemotherapy agent produced by the plant

Medium-chain alcohol dehydrogenases (ADHs) comprise a highly conserved enzyme family that catalyse the reversible reduction of aldehydes. However, recent discoveries in plant natural product biosynthesis suggest that the catalytic repertoire of ADHs has been expanded. Here we report the crystal structure of dihydroprecondylocarpine acetate synthase (DPAS), an ADH that catalyses the non-canonical 1,4-reduction of an α,β-unsaturated iminium moiety. Comparison with structures of plant-derived ADHs suggest the 1,4-iminium reduction does not require a proton relay or the presence of a catalytic zinc ion in contrast to canonical 1,2-aldehyde reducing ADHs that require the catalytic zinc and a proton relay. Furthermore, ADHs that catalysed 1,2-iminium reduction required the presence of the catalytic zinc and the loss of the proton relay. This suggests how the ADH active site can be modified to perform atypical carbonyl reductions, providing insight into how chemical reactions are diversified in plant metabolism.

Nature uses cycloaddition reactions to generate complex natural product scaffolds. Dehydrosecodine is a highly reactive biosynthetic intermediate that undergoes cycloaddition to generate several alkaloid scaffolds that are the precursors to pharmacologically important compounds such as vinblastine and ibogaine. Here we report how dehydrosecodine can be subjected to redox chemistry, which in turn allows cycloaddition reactions with alternative regioselectivity. By incubating dehydrosecodine with reductase and oxidase biosynthetic enzymes that act upstream in the pathway, we can access the rare pseudo-aspidosperma alkaloids, pseudo-tabersonine and pseudo-vincadifformine, both in vitro and by reconstitution in the plant Nicotiana benthamiana from an upstream intermediate. We propose a stepwise mechanism to explain the formation of the pseudo-tabersonine scaffold by structurally characterizing enzyme intermediates, and by monitoring the incorporation of deuterium labels. This discovery highlights how plants use redox enzymes to enantioselectively generate new scaffolds from common precursors.

Iridoid monoterpenes, widely distributed in plants and insects, have many ecological functions. While the biosynthesis of iridoids has been extensively studied in plants, little is known about how insects synthesize these natural products. Here, we elucidated the biosynthesis of the iridoids cis-trans-nepetalactol and cis-trans-nepetalactone in the pea aphid Acyrthosiphon pisum [Harris], where they act as sex pheromones. The exclusive production of iridoids in hind legs of sexual female aphids allowed us to identify iridoid genes by searching for genes specifically expressed in this tissue. Biochemical characterization of candidate enzymes revealed that the iridoid pathway in aphids proceeds through the same sequence of intermediates as described for plants. The six identified aphid enzymes are unrelated to their counterparts in plants, conclusively demonstrating an independent evolution of the entire iridoid pathway in plants and insects. In contrast to the plant pathway, at least three of the aphid iridoid enzymes are likely membrane-bound. We demonstrated that a lipid environment facilitates the cyclization of a reactive enol intermediate to the iridoid cyclopentanoid-pyran scaffold in vitro, suggesting that membranes are an essential component of the aphid iridoid pathway. Altogether, our discovery of this complex insect metabolic pathway establishes the genetic and biochemical basis for the formation of iridoid sex pheromones in aphids and this discovery also serves as a foundation for understanding the convergent evolution of complex metabolic pathways between kingdoms.Significance StatementPlants, animals and microbes produce a plethora of natural products that are important for defense and communication. Most of these compounds show a phylogenetically restricted occurrence, but in rare instances, the same natural product is biosynthesized by organisms in two different kingdoms. The monoterpene-derived iridoids, for example, have been found in more than 50 plant families, but are also observed in several insect orders. The aphid iridoid pathway discovered in this study, one of the longest and most chemically complex insect-derived natural product biosynthetic pathways reported to date, is compared with iridoid biosynthetic pathways in plants and highlights the mechanisms underlying the convergent evolution of metabolic enzymes in insects and plants.

This paper studies the influence of zinc coating of galvanized DP600 steel (DP600G) on nugget development and on the mechanical properties of dissimilar DP600 - AISI304 welded joints obtained by resistance spot welding process (RSW). RSW evaluations consisted of determining, from the dynamic resistance curves, the different stages involved in nugget formation. The experimental results showed that zinc coating on the DP600G steel has a significant influence on the time needed for the start of nugget formation, producing a two times longer delay for non-galvanized steel. Expulsion time was delayed by 1.12 times. The presence of Zn in DP600G/AISI304 welded joints produced smaller nugget diameters as compared to the DP600/AISI304 joints for the same experimental conditions; however, higher peak failure load values were obtained for the former. The causes of this unusual behavior are also analyzed in this paper. Resumen Este trabajo investiga la influencia del recubrimiento de zinc del acero DP600 (DP600G) en la formación del punto y las propiedades mecánicas de las uniones disímiles DP600 - AISI304 obtenidas mediante el proceso de soldadura por resistencia eléctrica por puntos (RSW). El análisis consistió en determinar, a partir de las curvas de resistencia dinámica, las diferentes etapas en la formación del punto. Los resultados experimentales mostraron que el recubrimiento de zinc del acero DP600G influye significativamente en el tiempo necesario para el inicio de la formación del punto, dos veces superior al del acero no galvanizado. El tiempo de la expulsión se retrasó 1,12 veces. La presencia de Zn en las uniones soldadas DP600G/AISI304 produjo diámetros del punto más pequeños en comparación con las uniones DP600/AISI304 para las mismas condiciones experimentales; sin embargo, se obtuvieron valores de carga máxima de fallo más altos para las primeras.

Thousands of natural products are derived from the fused cyclopentane-pyran molecular scaffold nepetalactol. These natural products are used in an enormous range of applications that span the agricultural and medical industries. For example, nepetalactone, the oxidized derivative of nepetalactol, is known for its cat attractant properties as well as potential as an insect repellent. Most of these naturally occurring nepetalactol-derived compounds arise from only two out of the eight possible stereoisomers, 7S-cis-trans and 7R-cis-cis nepetalactols. Here we use a combination of naturally occurring and engineered enzymes to produce seven of the eight possible nepetalactol or nepetalactone stereoisomers. These enzymes open the possibilities for biocatalytic production of a broader range of iridoids, providing a versatile system for the diversification of this important natural product scaffold.

Monoterpene indole alkaloids (MIAs) are a diverse class of plant natural products that include a number of medicinally important compounds. We set out to reconstitute the pathway for strictosidine, a key intermediate of all MIAs, from central metabolism in Nicotiana benthamiana. A disadvantage of this host is that its rich background metabolism results in the derivatization of some heterologously produced molecules. Here we use transcriptomic analysis to identify glycosyltransferases that are upregulated in response to biosynthetic intermediates and produce plant lines with targeted mutations in the genes encoding them. Expression of the early MIA pathway in these lines produces a more favorable product profile. Strictosidine biosynthesis was successfully reconstituted, with the best yields obtained by the co-expression of 14 enzymes, of which a major latex protein-like enzyme (MLPL) from Nepeta (catmint) is critical for improving flux through the iridoid pathway. The removal of endogenous glycosyltransferases does not impact the yields of strictosidine, highlighting that the metabolic flux of the pathway enzymes to a stable biosynthetic intermediate minimizes the need to engineer the endogenous metabolism of the host. The production of strictosidine in planta expands the range of MIA products amenable to biological synthesis.

Advances in omics technologies now permit generation of highly contiguous genome assemblies, detection of transcripts and metabolites at the level of single cells, and high-resolution determination of gene regulatory features including 3-dimensional chromatin interactions. Using a complementary, multi-omics approach, we interrogated the monoterpene indole alkaloid (MIA) biosynthetic pathway in Catharanthus roseus, a source of leading anti-cancer drugs. We identified not only new clusters of genes involved in MIA biosynthesis on the eight C. roseus chromosomes but also rampant gene duplication including paralogs of MIA pathway genes. Clustering was not limited to the linear genome and through chromatin interaction data, MIA pathway genes were shown to be present within the same topologically associated domain, permitting identification of a secologanin transporter. Single cell RNA-sequencing revealed exquisite and sequential cell-type specific partitioning of the leaf MIA biosynthetic pathway that, when coupled with a newly developed single cell metabolomics approach, permitted identification of a reductase that yields the bis-indole alkaloid anhydrovinblastine. Last, we revealed cell-type specific expression in the root MIA pathway that is conferred in part by neo- and sub-functionalization of paralogous MIA pathway genes. This study highlights how a suite of omic approaches, including single cell gene expression and metabolomics, can efficiently link sequence with function in complex, specialized metabolic pathways of plants.

Plants produce a wide variety of pharmacologically active molecules classified as natural products. Derivatization of these natural products can modulate or improve the bioactivity of the parent compound. Unfortunately, chemical derivatization of natural products is often difficult or impractical. Here we use the newly discovered biosynthetic genes for two monoterpene indole alkaloids, alstonine and stemmadenine acetate, to generate analogs of these compounds. We reconstitute these biosynthetic genes in the heterologous host Nicotiana benthamiana along with an unnatural starting substrate to produce the corresponding new-to-nature alkaloid product.

Plants produce a wide variety of pharmacologically active molecules classified as natural products. Derivatization of these natural products can modulate or improve the bioactivity of the parent compound. Unfortunately, chemical derivatization of natural products is often difficult or impractical. Here we use the newly discovered biosynthetic genes for two monoterpene indole alkaloids, alstonine and stemmadenine acetate, to generate analogs of these compounds. We reconstitute these biosynthetic genes in the heterologous host

Thousands of natural products are derived from the fused cyclopentane-pyran molecular scaffold nepetalactol. These natural products are used in an enormous range of applications that span the agricultural and medical industries. For example, nepetalactone, the oxidized derivative of nepetalactol, is known for its cat attractant properties as well as potential as an insect repellent. Most of these naturally occurring nepetalactol-derived compounds arise from only two out of the eight possible stereoisomers, 7S-cis-trans and 7R-cis-cis nepetalactols. Here we use a combination of naturally occurring and engineered enzymes to produce seven of the eight possible nepetalactol or nepetalactone stereoisomers. These enzymes open the possibilities for biocatalytic production of a broader range of iridoids, providing a versatile system for the diversification of this important natural product scaffold.

Specialized metabolites are chemically complex small molecules with a myriad of biological functions. To investigate plant-specialized metabolite biosynthesis more effectively, we developed an improved method for virus-induced gene silencing (VIGS). We designed a plasmid that incorporates fragments of both the target gene and knockdown marker gene (phytoene desaturase, PDS), which identifies tissues that have been successfully silenced in planta. To demonstrate the utility of this method, we used the terpenoid indole alkaloid (TIA) pathway in Madagascar periwinkle (Catharanthus roseus) as a model system. Catharanthus roseus is a medicinal plant well known for producing many bioactive compounds, such as vinblastine and vincristine. Our VIGS method enabled the discovery of a previously unknown biosynthetic enzyme, serpentine synthase (SS). This enzyme is a cytochrome P450 (CYP) that produces the β-carboline alkaloids serpentine and alstonine, compounds with strong blue autofluorescence and potential pharmacological activity. The discovery of this enzyme highlights the complexity of TIA biosynthesis and demonstrates the utility of this improved VIGS method for discovering unidentified metabolic enzymes in plants., An improved virus-induced gene silencing approach led to the discovery of the alkaloid biosynthetic enzyme serpentine synthase.

Authors gratefully acknowledge the Max Planck Society, ERA-IB project NBCPBH-EIB.13.008 (SO’C and RG) and ERC 788301 (SO’C). Plant secondary metabolites have applications for the food, biofuel, and pharmaceutical industries. Recent advances in pathway elucidation and host expression systems now allow metabolic engineering of plant metabolic pathways to produce “new-to-nature” derivatives with novel biological activities, thereby amplifying the range of industrial uses for plant metabolites. Here we use a transient expression system in the model plant Nicotiana benthamiana to reconstitute the two-step plant-derived biosynthetic pathway for auxin (indole acetic acid) to achieve accumulation up to 500 ng/g fresh mass (FM). By expressing these plant-derived enzymes in combination with either bacterial halogenases and alternative substrates, we can produce both natural and new-to-nature halogenated auxin derivatives up to 990 ng/g FM. Proteins from the auxin synthesis pathway, tryptophan aminotransferases (TARs) and flavin-dependent monooxygenases (YUCs), could be transiently expressed in combination with four separate bacterial halogenases to generate halogenated auxin derivatives. Brominated auxin derivatives could also be observed after infiltration of the transfected N. benthamiana with potassium bromide and the halogenases. Finally, the production of additional auxin derivatives could also be achieved by co-infiltration of TAR and YUC genes with various tryptophan analogs. Given the emerging importance of transient expression in N. benthamiana for industrial scale protein and product expression, this work provides insight into the capacity of N. benthamiana to interface bacterial genes and synthetic substrates to produce novel halogenated metabolites. Publisher PDF

Plant hormones have various functions in plants and play crucial roles in all developmental and differentiation stages. Auxins constitute one of the most important groups with the major representative indole-3-acetic acid (IAA). A halogenated derivate of IAA, 4-chloro-indole-3-acetic acid (4-Cl-IAA), has previously been identified in Pisum sativum and other legumes. While the enzymes responsible for the halogenation of compounds in bacteria and fungi are well studied, the metabolic pathways leading to the production of 4-Cl-IAA in plants, especially the halogenating reaction, are still unknown. Therefore, bacterial flavin-dependent tryptophan-halogenase genes were transformed into the model organism Arabidopsis thaliana. The type of chlorinated indole derivatives that could be expected was determined by incubating wild type A. thaliana with different Cl-tryptophan derivatives. We showed that, in addition to chlorinated IAA, chlorinated IAA conjugates were synthesized. Concomitantly, we found that an auxin conjugate synthetase (GH3.3 protein) from A. thaliana was able to convert chlorinated IAAs to amino acid conjugates in vitro. In addition, we showed that the production of halogenated tryptophan (Trp), indole-3-acetonitrile (IAN) and IAA is possible in transgenic A. thaliana in planta with the help of the bacterial halogenating enzymes. Furthermore, it was investigated if there is an effect (i) of exogenously applied Cl-IAA and Cl-Trp and (ii) of endogenously chlorinated substances on the growth phenotype of the plants.

Plant hormones have various functions in plants and play crucial roles in all developmental and differentiation stages. Auxins constitute one of the most important groups with the major representative indole-3-acetic acid (IAA). A halogenated derivate of IAA, 4-chloro-indole-3-acetic acid (4-Cl-IAA), has previously been identified in Pisum sativum and other legumes. While the enzymes responsible for the halogenation of compounds in bacteria and fungi are well studied, the metabolic pathways leading to the production of 4-Cl-IAA in plants, especially the halogenating reaction, are still unknown. Therefore, bacterial flavin-dependent tryptophan-halogenase genes were transformed into the model organism Arabidopsis thaliana. The type of chlorinated indole derivatives that could be expected was determined by incubating wild type A. thaliana with different Cl-tryptophan derivatives. We showed that, in addition to chlorinated IAA, chlorinated IAA conjugates were synthesized. Concomitantly, we found that an auxin conjugate synthetase (GH3.3 protein) from A. thaliana was able to convert chlorinated IAAs to amino acid conjugates in vitro. In addition, we showed that the production of halogenated tryptophan (Trp), indole-3-acetonitrile (IAN) and IAA is possible in transgenic A. thaliana in planta with the help of the bacterial halogenating enzymes. Furthermore, it was investigated if there is an effect (i) of exogenously applied Cl-IAA and Cl-Trp and (ii) of endogenously chlorinated substances on the growth phenotype of the plants.

Plant monoterpene indole alkaloids, a large class of natural products, derive from the biosynthetic intermediate strictosidine aglycone. Strictosidine aglycone, which can exist as a variety of isomers, can be reduced to form numerous different structures. We have discovered a short-chain alcohol dehydrogenase (SDR) from plant producers of monoterpene indole alkaloids (Catharanthus roseus and Rauvolfia serpentina) that reduce strictosidine aglycone and produce an alkaloid that does not correspond to any previously reported compound. Here we report the structural characterization of this product, which we have named vitrosamine, as well as the crystal structure of the SDR. This discovery highlights the structural versatility of the strictosidine aglycone biosynthetic intermediate and expands the range of enzymatic reactions that SDRs can catalyse. This discovery further highlights how a sequence-based gene mining discovery approach in plants can reveal cryptic chemistry that would not be uncovered by classical natural product chemistry approaches.

During the last years halogenated compounds have drawn a lot of attention. Metabolites with one or more halogen atoms are often more active than their non-halogenated derivatives like indole-3-acetic acid (IAA) and 4-Cl-IAA. Within this work, bacterial flavin-dependent tryptophan halogenase genes were inserted into Brassica rapa ssp. pekinensis (Chinese cabbage) with the aim to produce novel halogenated indole compounds. It was investigated which tryptophan-derived indole metabolites, such as indole glucosinolates or potential degradation products can be synthesized by the transgenic root cultures. In vivo and in vitro activity of halogenases heterologously produced was shown and the production of chlorinated tryptophan in transgenic root lines was confirmed. Furthermore, chlorinated indole-3-acetonitrile (Cl-IAN) was detected. Other tryptophan-derived indole metabolites, such as IAA or indole glucosinolates were not found in the transgenic roots in a chlorinated form. The influence of altered growth conditions on the amount of produced chlorinated compounds was evaluated. We found an increase in Cl-IAN production at low temperatures (8 °C), but otherwise no significant changes were observed. Furthermore, we were able to regenerate the wild type and transgenic root cultures to adult plants, of which the latter still produced chlorinated metabolites. Therefore, we conclude that the genetic information had been stably integrated. The transgenic plants showed a slightly altered phenotype compared to plants grown from seeds since they also still expressed the rol genes. By this approach we were able to generate various stably transformed plant materials from which it was possible to isolate chlorinated tryptophan and Cl-IAN.

Cycloaddition reactions generate chemical complexity in a single step. Here we report the crystal structures of three homologous plant-derived cyclases involved in the biosynthesis of iboga and aspidosperma alkaloids. These enzymes act on the same substrate, named angryline, to generate three distinct scaffolds. Mutational analysis reveals how these highly similar enzymes control regio- and stereo-selectivity.

Catharanthus roseus is a medicinal plant well known for producing bioactive compounds such as vinblastine and vincristine, which are classified as terpenoid indole alkaloids (TIAs). Although the leaves of this plant are the main source of these antitumour drugs, much remains unknown on how TIAs are biosynthesised from a central precursor, strictosidine, to various TIAs in planta. Here, we have succeeded in showing, for the first time in leaf tissue of C. roseus, cell-specific TIAs localisation and accumulation with 10 μm spatial resolution Imaging mass spectrometry (Imaging MS) and live single-cell mass spectrometry (single-cell MS). These metabolomic studies revealed that most TIA precursors (iridoids) are localised in the epidermal cells, but major TIAs including serpentine and vindoline are localised instead in idioblast cells. Interestingly, the central TIA intermediate strictosidine also accumulates in both epidermal and idioblast cells of C. roseus. Moreover, we also found that vindoline accumulation increases in laticifer cells as the leaf expands. These discoveries highlight the complexity of intercellular localisation in plant specialised metabolism.

How to make bioactive alkaloids Vinblastine and vincristine are important, expensive anticancer agents that are produced by dimerization of the plant-derived alkaloids catharanthine and vindoline. The enzymes that transform tabersonine into vindoline are known; however, the mechanism by which the scaffolds of catharanthine and tabersonine are generated has been a mystery. Caputi et al. now describe the biosynthetic genes and corresponding enzymes responsible. This resolves a long-standing question of how plant alkaloid scaffolds are synthesized, which is important not only for vinblastine and vincristine biosynthesis, but also for understanding the many other biologically active alkaloids found throughout nature. Science , this issue p. 1235

Levansucrases are members of the glycoside hydrolase family and catalyse both the hydrolysis of the substrate sucrose and the transfer of fructosyl units to acceptor molecules. In the presence of sufficient sucrose, this may either lead to the production of fructooligosaccharides or fructose polymers. Aim of this study is to rationalise the differences in the polymerisation properties of bacterial levansucrases and in particular to identify structural features that determine different product spectrum in the levansucrase of the Gram-negative bacterium Erwinia amylovora (Ea Lsc, EC 2.4.1.10) as compared to Gram-positive bacteria such as Bacillus subtilis levansucrase. Ea is an enterobacterial pathogen responsible for the Fire Blight disease in rosaceous plants (e.g., apple and pear) with considerable interest for the agricultural industry. The crystal structure of Ea Lsc was solved at 2.77 Å resolution and compared to those of other fructosyltransferases from Gram-positive and Gram-negative bacteria. We propose the structural features, determining the different reaction products, to reside in just a few loops at the rim of the active site funnel. Moreover we propose that loop 8 may have a role in product length determination in Gluconacetobacter diazotrophicus LsdA and Microbacterium saccharophilum FFase. The Ea Lsc structure shows for the first time the products of sucrose hydrolysis still bound in the active site.

Summary The extraordinary chemical diversity of the plant-derived monoterpene indole alkaloids, which include vinblastine, quinine, and strychnine, originates from a single biosynthetic intermediate, strictosidine aglycone. Here we report for the first time the cloning of a biosynthetic gene and characterization of the corresponding enzyme that acts at this crucial branchpoint. This enzyme, an alcohol dehydrogenase homolog, converts strictosidine aglycone to the heteroyohimbine-type alkaloid tetrahydroalstonine. We also demonstrate how this enzyme, which uses a highly reactive substrate, may interact with the upstream enzyme of the pathway., Graphical Abstract, Highlights • Tetrahydroalstonine synthase catalyzes the formation of a plant-derived alkaloid • Tetrahydroalstonine synthase is localized to the nucleus • Tetrahydroalstonine synthase and the preceding pathway enzyme interact • Discovery of a gene controlling structural diversity of monoterpene indole alkaloids, How plants transform the central biosynthetic intermediate strictosidine into thousands of divergent alkaloids has remained unresolved. Stavrinides et al. discover a nuclear-localized alcohol dehydrogenase homolog responsible for conversion of strictosidine aglycone to tetrahydroalstonine that appears to interact with an upstream pathway enzyme.

Nepetalactones are iridoid monoterpenes with a broad range of biological activities produced by plants in the Nepeta genus. However, none of the genes for nepetalactone biosynthesis have been discovered. Here we report the transcriptomes of two Nepeta species, each with distinctive profiles of nepetalactone stereoisomers. As a starting point for investigation of nepetalactone biosynthesis in Nepeta, these transcriptomes were used to identify candidate genes for iridoid synthase homologs, an enzyme that has been shown to form the core iridoid skeleton in several iridoid producing plant species. Iridoid synthase homologs identified from the transcriptomes were cloned, heterologously expressed, and then assayed with the 8-oxogeranial substrate. These experiments revealed that catalytically active iridoid synthase enzymes are present in Nepeta, though there are unusual mutations in key active site residues. Nevertheless, these enzymes exhibit similar catalytic activity and product profile compared to previously reported iridoid synthases from other plants. Notably, four nepetalactone stereoisomers with differing stereochemistry at the 4α and 7α positions – which are generated during the iridoid synthase reaction – are observed at different ratios in various Nepeta species. This work strongly suggests that the variable stereochemistry at these 4α and 7α positions of nepetalactone diastereomers is established further downstream in the iridoid pathway in Nepeta. Overall, this work provides a gateway into the biosynthesis of nepetalactones in Nepeta., Graphical abstract Iridoid synthase from Nepeta cateria (catnip) and Nepeta mussinii, have been cloned and characterized.Image 1, Highlights • Species within the Nepeta genus (such as catnip) produce nepetalactone iridoids. • The enzymes that produce the iridoid scaffold of nepetalactone were identified from two species of Nepeta. • The iridoid synthase enzymes are not responsible for the stereochemical variation in these iridoids.

The Gram-negative bacteriumErwinia amylovorais a destructive pathogen of plants belonging to the Rosaceae family. Amongst its pathogenicity factors,E. amylovoraproduces the exopolysaccharide amylovoran, which contributes to the occlusion of plant vessels, causing wilting of shoots and eventually resulting in plant death. Amylovoran biosynthesis requires the presence of 12 genes (fromamsA toamsL) clustered in theamsregion of theE. amylovoragenome. They mostly encode glycosyl transferases (AmsG, AmsB, AmsD, AmsE, AmsJ and AmsK), proteins involved in amylovoran translocation and assembly (AmsH, AmsL and AmsC), and also a tyrosine kinase (AmsA) and a tyrosine phosphatase (AmsI), which are both involved in the regulation of amylovoran biosynthesis. The low-molecular-weight protein tyrosine phosphatase AmsI was overexpressed as a His6-tagged protein inEscherichia coli, purified and crystallized. X-ray diffraction data were collected to a maximum resolution of 1.57 Å in space groupP3121.

The carbon skeleton of ecologically and pharmacologically important iridoid monoterpenes is formed in a reductive cyclization reaction unrelated to canonical terpene cyclization. Here we report the crystal structure of the recently discovered iridoid cyclase (from Catharanthus roseus) bound to a mechanism-inspired inhibitor that illuminates substrate binding and catalytic function of the enzyme. Key features that distinguish iridoid synthase from its close homolog progesterone 5 beta-reductase are highlighted.

Acta crystallographica / F 69(5), 570 - 573 (2013). doi:10.1107/S1744309113010750, Published by Blackwell, Oxford [u.a.]

*† These authors equally contributed to this work. SUMMARY For almost a decade, our knowledge on the organisation of the family 1 UDP-glycosyltransferases (UGTs) has been limited to the model plant A. thaliana. The availability of other plant genomes represents an opportunity to obtain a broader view of the family in terms of evolution and organisation. Family 1 UGTs are known to glycosylate several classes of plant secondary metabolites. A phylogeny reconstruction study was performed to get an insight into the evolution of this multigene family during the adaptation of plants to life on land. The organisation of the UGTs in the different organisms was also investigated. More than 1500 putative UGTs were identified in 12 fully sequenced and assembled plant genomes based on the highly conserved PSPG motif. Analyses by maximum likelihood (ML) method were performed to reconstruct the phylogenetic relationships existing between the sequences. The results of this study clearly show that the UGT family expanded during the transition from algae to vascular plants and that in higher plants the clustering of UGTs into phylogenetic groups appears to be conserved, although gene loss and gene gain events seem to have occurred in certain lineages. Interestingly, two new phylogenetic groups, named O and P, that are not present in A. thaliana were discovered.

Biosynthesis of the sesquiterpene rotundone in Vespolina grapes during berry ripening was investigated over two consecutive seasons, revealing that the compound accumulates from veraison to harvest and reaches relatively high concentrations (up to 5.44 μg/kg). Rotundone levels up to 1.91 μg/kg were also found in clones of Gruener Veltliner, a white grape variety known to give 'peppery' wines. These concentrations are higher than those reported for Syrah grapes and are similar to the levels found in some plants. Rotundone was shown to accumulate almost exclusively in berry exocarp, suggesting that skin contact during winemaking could be used to modulate the peppery character of red wine. However, rotundone yield after the winemaking process was relatively low. Indeed, only 10% of the rotundone present in grapes was extracted during fermentation, and only 6% was recovered in bottled wine. The results presented in this work provide key knowledge for manipulation of the peppery character of wine in order to optimize the intensity of this characteristic wine aroma.

Optimal solvent conditions for grape sample preparation were investigated for the purpose of metabolite profiling studies, with the aim of obtaining as many features as possible with the best analytical repeatability. Mixtures of water, methanol and chloroform in different combinations were studied as solvents for the extraction of ground grapes. The experimental design used a two stage study to find the optimum extraction medium. The extracts obtained were further purified using solid phase extraction and analysed using a UPLC full scan TOF MS with both reversed phase and hydrophilic interaction chromatography. The data obtained were processed using data extraction algorithms and advanced statistical software for data mining. The results obtained indicated that a fairly broad optimal area for solvent composition could be identified, containing approximately equal amounts of methanol and chloroform and up to 20% water. Since the water content of the samples was variable, the robustness of the optimal conditions suggests these are appropriate for large scale profiling studies for characterisation of the grape metabolome.

Rotundone is an oxygenated sesquiterpene belonging to the family of guaianes, giving the ‘peppery’ aroma to white and black pepper and to red wines. Here we describe a novel, convenient protocol for the synthesis of rotundone, starting from a commercially available compound and requiring only two reaction steps, and an improved, faster method of GC separation (30 min) with selective quantisation of rotundone using tandem mass spectrometry in multiple reaction monitoring (MRM) mode with d5-rotundone as internal standard. With limits of detection (LODs) of 1.5 ng/L in white wine and 2.0 ng/L in red wine, intraday repeatability CV values of 6% and 5% at 50 ng/L and 500 ng/L and interday repeatability CV values of 13% and 6% at 50 ng/L and 500 ng/L, respectively, the improved protocol provides the desired sensitivity and selectivity for routine analysis of rotundone in both white and red wines. Initial application of this method highlighted the presence of unexpectedly high concentrations of rotundone, thus explaining the origin of the distinctive peppery aroma in Schioppettino and Vespolina red wines and in Gruener Veltliner white wines. Copyright © 2011 John Wiley & Sons, Ltd.

The synthesis of terpenoid glycosides typically uses a chemical strategy since few biocatalysts have been identified that recognise these scaffolds. In this study, a platform of 107 recombinant glycosyltransferases (GTs), comprising the multigene family of small molecule GTs of Arabidopsis thaliana have been screened against a range of model terpenoid acceptors to identify those enzymes with high activity. Twenty-seven GTs are shown to glycosylate a diversity of mono-, sesqui- and diterpenes, such as geraniol, perillyl alcohol, artemisinic acid and retinoic acid. Certain enzymes showing substantial sequence similarity recognise terpenoids containing a primary alcohol, irrespective of the linear or cyclical structure of the scaffold; other GTs glycosylate scaffolds containing secondary and tertiary alcohols; the carboxyl group of other terpenoids also represents a feature that is recognized by GTs previously known to form glucose esters with many different compounds. These data underpin the rapid prediction of potential biocatalysts from GT sequence information. To explore the potential of GTs as biocatalysts, their use for the production of terpenoid glycosides was investigated by using a microbial-based whole-cell biotransformation system capable of regenerating the cofactor, UDP-glucose. A high cell density fermentation system was shown to produce several hundred milligrams of a model terpenoid, geranyl-glucoside. The activities of the GTs are discussed in relation to their substrate recognition and their utility in biotransformations as a complement or alternative to chemical synthesis.

Plants produce an enormous array of biologically active metabolites, often with stereochemical variations on the same molecular scaffold. These changes in stereochemistry dramatically impact biological activity. Notably, the stereoisomers of the heteroyohimbine alkaloids show diverse pharmacological activities. We reported a medium chain dehydrogenase/reductase (MDR) from Catharanthus roseus that catalyses formation of a heteroyohimbine isomer. Here we report the discovery of additional heteroyohimbine synthases (HYSs), one of which produces a mixture of diastereomers. The crystal structures for three HYSs have been solved, providing insight into the mechanism of reactivity and stereoselectivity, with mutation of one loop transforming product specificity. Localization and gene silencing experiments provide a basis for understanding the function of these enzymes in vivo. This work sets the stage to explore how MDRs evolved to generate structural and biological diversity in specialized plant metabolism and opens the possibility for metabolic engineering of new compounds based on this scaffold., The stereochemistry of the plant heteroyohimbine alkaloids is a key factor determining their diverse biological activities. Here, the authors carry out structural, localization and genetic experiments to understand the mechanism of stereoselectivity for three heteroyohimbine synthases and to identify their function in vivo.

Microperoxidases are small heme-peptides obtained by proteolytic digestion of cytochrome c, exhibiting peroxidase activity. They consist of a short- or medium-length polypeptide chain, covalently linked to an iron protoporphyrin IX moiety via two thioether bonds involving Cys residues at the c-porphyrin A and B pyrrole rings. These small molecules are interesting for a wide range of possible applications. We have structurally characterized, by means of electrospray ionization (ESI) mass and tandem mass spectrometric experiments, a novel microperoxidase called MMP-5 (Marinobacter MicroPeroxidase-5), obtained by proteolytic digestion of cytochrome c552, a monoheminic electron-transfer protein isolated from Marinobacter hydrocarbonoclasticus. This microperoxidase, which still maintains the functional peptide moieties for peroxidase activity, is devoid of the two amino acids intercalating the Cys residues linked to the c-porphyrin, thus increasing its water solubility. Once submitted to the ESI source potential, MMP-5 showed an interesting tendency for the reduction of the iron protoporphyrin substructure. This behaviour was clearly evidenced by the mass shift exhibited by the reduced form. Copyright © 2005 John Wiley & Sons, Ltd.

The ergot alkaloids, a class of fungal‐derived natural products with important biological activities, are derived from a common intermediate, chanoclavine‐I, which is elaborated into a set of diverse structures. Herein we report the discovery of the biosynthetic pathway of cycloclavine, a complex ergot alkaloid containing a cyclopropyl moiety. We used a yeast‐based expression platform along with in vitro biochemical experiments to identify the enzyme that catalyzes a rearrangement of the chanoclavine‐I intermediate to form a cyclopropyl moiety. The resulting compound, cycloclavine, was produced in yeast at titers of >500 mg L−1, thus demonstrating the feasibility of the heterologous expression of these complex alkaloids.

Erwinia amylovora is a plant pathogen that affects Rosaceae, such as apple and pear. In E. amylovora the fructans, produced by the action of a levansucrase (EaLsc), play a role in virulence and biofilm formation. Fructans are bioactive compounds, displaying health-promoting properties in their own right. Their use as food and feed supplements is increasing. In this study, we investigated the biomolecular properties of EaLsc using HPAEC-PAD, MALDI-TOF MS, and spectrophotometric assays. The enzyme, which was heterologously expressed in Escherichia coli in high yield, was shown to produce mainly fructooligosaccharides (FOSs) with a degree of polymerization between 3 and 6. The kinetic properties of EaLsc were similar to those of other phylogenetically related Gram-negative bacteria, but the good yield of FOSs, the product spectrum, and the straightforward production of the enzyme suggest that EaLsc is an interesting biocatalyst for future studies aimed at producing tailor-made fructans.

Graphical abstract, In connection with prospective 18F-PET imaging studies, the potential for enzymatic synthesis of fluorine-labelled glycosides of small molecules was investigated. Approaches to the enzymatic synthesis of anomeric phosphates of d-gluco-configured fluorosugars proved ineffective. In contrast, starting in the d-galacto series and relying on the consecutive action of Escherichia coli galactokinase (GalK), galactose-1-phosphate uridylyltransferase (GalPUT), uridine-5′-diphosphogalactose 4-epimerase (GalE) and oat root glucosyltransferase (SAD10), a quick and effective synthesis of 6-deoxy-6-fluoro-d-glucosyl N-methylanthranilate ester was achieved.

For almost a decade, our knowledge on the organisation of the family 1 UDP-glycosyltransferases (UGTs) has been limited to the model plant A. thaliana. The availability of other plant genomes represents an opportunity to obtain a broader view of the family in terms of evolution and organisation. Family 1 UGTs are known to glycosylate several classes of plant secondary metabolites. A phylogeny reconstruction study was performed to get an insight into the evolution of this multigene family during the adaptation of plants to life on land. The organisation of the UGTs in the different organisms was also investigated. More than 1500 putative UGTs were identified in 12 fully sequenced and assembled plant genomes based on the highly conserved PSPG motif. Analyses by maximum likelihood (ML) method were performed to reconstruct the phylogenetic relationships existing between the sequences. The results of this study clearly show that the UGT family expanded during the transition from algae to vascular plants and that in higher plants the clustering of UGTs into phylogenetic groups appears to be conserved, although gene loss and gene gain events seem to have occurred in certain lineages. Interestingly, two new phylogenetic groups, named O and P, that are not present in A. thaliana were discovered.

Terpenoids represent the oldest known biomolecules, having been recovered from sediments as old as 2.5 billion years. Among plant secondary metabolites, they are the most abundant and diverse class of natural compounds. The diversity of terpenoids is probably a reflection of their many biological activities in nature, which has made them a widely used resource for traditional and modern human exploitation. They are usually the main constituents of essential oils of most plants offering a wide variety of pleasant scents from flowery to fruity, to woody or balsamic notes. For this reason terpenoids constitute a very important class of compounds for flavour and fragrance industries, in fact, in the US alone, the demand is forecast to grow 3.7 percent per year to $5.3 billion in 2012. The recent patents on production and extraction of terpenoids commonly used as natural flavouring compounds in food industries are reviewed in the present manuscript.

Polymorphonuclear leukocyte infiltration and activation into colonic mucosa are believed to play a pivotal role in mediating tissue damage in human ulcerative colitis (UC). Ligands of human CXC chemokine receptor 1 and 2 (CXCR1/R2) are chemoattractants of PMN, and high levels were found in the mucosa of UC patients. To investigate the pathophysiological role played by CXCR2 in experimental UC, we induced chronic experimental colitis in WT and CXCR2−/− mice by two consecutive cycles of 4% dextran sulfate sodium administration in drinking water. In wild-type (WT) mice, the chronic relapsing of DSS-induced colitis was characterized by clinical signs and histopathological findings that closely resemble human disease. CXCR2−/− mice failed to show PMN infiltration into the mucosa and, consistently with a key role of PMN in mediating tissue damage in UC, showed limited signs of mucosal damage and reduced clinical symptoms. Our data demonstrate that CXCR2 plays a key pathophysiological role in experimental UC, suggesting that CXCR2 activation may represent a relevant pharmacological target for the design of novel pharmacological treatments in human UC.

The preparation and characterization of a new microperoxidase obtained from proteinase K-treated cytochrome c 552 from Marinobacter hydrocarbonoclasticus (previously known as Pseudomonas nautica ) are presented. This microperoxidase (MMP-5) has novel structural properties relative to previously reported microperoxidases, as the two intervening amino acid (X) residues within the consensual CXXCH c -type heme binding motif are missing, yielding a heme-pentapeptide with increased solubility in aqueous solvents and a 1–2 order of magnitude higher stability of the monomeric state relative to canonical microperoxidases. The electronic spectra in the near-UV and visible regions have been studied as a function of MMP-5 concentration and pH. The spectroscopic properties of MMP-5 are typical of microperoxidases with high-spin hexa- or pentacoordinate heme species dominant in the 1–8 pH range and low-spin states prevailing at higher pH values. In the presence of hydrogen peroxide, MMP-5 displays peroxidatic activities towards several compounds.