Shikimate Pathway Research Articles

Improved aromatic alcohol production by strengthening the shikimate pathway in Saccharomyces cerevisiae

Aromatic alcohols, such as tyrosol and 2-phenylethanol, are widely used in various industries owing to their superior flavors and properties. In this study, a yeast metabolic modification platform for the de novo synthesis of tyrosol and 2-phenylethanol was established. Alleviation of the feedback inhibition and overexpression of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase and chorismate mutase were achieved by introducing aroGD146N and pheAfbr genes from Escherichia coli. Shikimate kinase (aroL) and shikimate dehydrogenase (ydiB), analogues of the penta-functional enzyme Aro1p, were overexpressed to release the metabolic bottlenecks. Furthermore, in addition to ARO10-overexpression, ARO9 was disrupted to reduce the by-product generation. Finally, by introducing EcaroGD146N, EcpheAfbr, EcydiB and EcaroL in the ARO9-deleted strain, together with overexpression of transketolase (TKL1) and phenylpyruvate decarboxylase (ARO10), the engineered strain JM26 was constructed. Using glucose as the sole carbon source, JM26 achieved 541 mg/L (73.5 mg/g DCW) of tyrosol and 643 mg/L (92.0 mg/g DCW) of 2-phenylethanol, which is 5.7-fold and 6.1-fold higher than the control strain, respectively. These results demonstrate that the strategies applied improved de novo production of tyrosol and 2-phenylethanol, and showed potential for application as a metabolic chassis for other aromatic compounds in Saccharomyces cerevisiae and the exploration of their biological functions.

In vivo and in vitro evaluation of the effect of glyphosate (Roundup) on Toxoplasma gondii.

Apicoplast, a derived non-photosynthetic plastid, which is found in most Apicomplexa, provides essential functions to parasites. The shikimate pathway is localized in the plant chloroplast as a remarkable route for the survival of the Toxoplasma. In this study, in vivo and in vitro effects of glyphosate (Roundup, Herbicide), as an inhibitor of the enzyme, were evaluated on T. gondii. Tachyzoites of RH strain were incubated for 1.5h in various concentrations (1-128µg/ml) of glyphosate. The parasite was cultivated in the cell monolayer of the heLa cell, and then the cultures were exposed to various concentrations. To evaluate the therapeutic quality, 2 × 105 tachyzoites were intradermally inoculated into ten mice from each group. Four doses of the compound were daily administrated every 24h after inoculation due 10days continuously. Also, two other groups were assigned as the positive and negative control. In flow cytometry, the highest mortality rate was related to concentrations of 128 and 256μg/ml, 18.29% and 18.64%, respectively, while the mortality rate was 0.03% in the negative control (P value > 0.05). Based on microscopic observation of the stained touch smear of the liver, all treated mice were killed by the parasite. This compound also had no lethal effect on the mice. According to the results of this study, glyphosate is not a good candidate for the treatment of toxoplasmosis. It seems that the parasite has another pathway for providing the essential amino acids.

Adaptation of bacteria to glyphosate: a microevolutionary perspective of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase.

Glyphosate is the leading herbicide worldwide, but it also affects prokaryotes because it targets the central enzyme (5-enolpyruvylshikimate-3-phosphate, EPSP) of the shikimate pathway in the synthesis of the three essential aromatic amino acids in bacteria, fungi and plants. Our results reveal that bacteria may easily become resistant to glyphosate through changes in the 5-enolpyruvylshikimate-3-phosphate synthase active site. This indicates the importance of examining how glyphosate affects microbe-mediated ecosystem functions and human microbiomes.

Optimization of Plant Extract Purification Procedure for Rapid Screening Analysis of Sixteen Phenolics by Liquid Chromatography

A solid-phase extraction (SPE) procedure was developed for simultaneous monitoring of sixteen different phenolics of various polarity, quantified by high-performance liquid chromatography (HPLC). The procedure allowed screening the accumulation of intermediates in different metabolic pathways that play a crucial role in plant physiology and/or are beneficial for human health. Metabolites mostly involved in phenylpropanoid, shikimate, and polyketide pathways comprise chlorogenic acid, gentisic acid, vanillic acid, caffeic acid, protocatechuic acid, ferulic acid, rutin, quercetin, epicatechin, gallic acid, sinapic acid, p-coumaric acid, o-coumaric acid, vanillin; two rarely quantified metabolites, 2,5-dimethoxybenzoic acid and 4-methoxycinnamic acid, were included as well. The procedure offered low cost, good overall efficiency, and applicability in laboratories with standard laboratory equipment. SPE recoveries were up to 99.8% at various concentration levels. The method allowed for routine analysis of compounds with a wide range of polarity within a single run, while its applicability was demonstrated for various model plant species (tobacco, wheat, and soybean), as well as different tissue types (shoots and roots).

Engineering a Synthetic Pathway for Gentisate in Pseudomonas Chlororaphis P3.

Pseudomonas chlororaphis P3 has been well-engineered as a platform organism for biologicals production due to enhanced shikimate pathway and excellent physiological and genetic characteristics. Gentisate displays high antiradical and antioxidant activities and is an important intermediate that can be used as a precursor for drugs. Herein, a plasmid-free biosynthetic pathway of gentisate was constructed by connecting the endogenous degradation pathway from 3-hydroxybenzoate in Pseudomonas for the first time. As a result, the production of gentisate reached 365 mg/L from 3-HBA via blocking gentisate conversion and enhancing the gentisate precursors supply through the overexpression of the rate-limiting step. With a close-up at the future perspectives, a series of bioactive compounds could be achieved by constructing synthetic pathways in conventional Pseudomonas to establish a cell factory.

Induction of tocopherol biosynthesis through heat shock treatment in Arabidopsis.

Plants have developed various self-defense systems to survive many types of unfavorable conditions. Heat shock (HS) treatment, an abiotic stress, activates salicylic acid (SA) biosynthesis to enhance resistance to biotic stresses in some plant species. Since SA is produced from the shikimate pathway, other related metabolic pathways were expected to be upregulated by HS treatment. We speculated that tocopherol biosynthesis utilizing chorismic acid would be activated by HS treatment. In Arabidopsis, expression analysis of tocopherol biosynthetic genes, HPPD, VTE2, VTE3, VTE1, and VTE4, in combination with measurement of metabolites, indicated that HS treatment enhanced the biosynthesis and accumulation of tocopherols. Analyses using an SA biosynthesis-deficient mutant indicated that the upregulation of tocopherol biosynthesis was independent of the SA-mediated signaling pathway.

Enhanced Production of Aromatic Amino Acids in Tobacco Plants Leads to Increased Phenylpropanoid Metabolites and Tolerance to Stresses.

Aromatic amino acids (AAAs) synthesized in plants via the shikimate pathway can serve as precursors for a wide range of secondary metabolites that are important for plant defense. The goals of the current study were to test the effect of increased AAAs on primary and secondary metabolic profiles and to reveal whether these plants are more tolerant to abiotic stresses (oxidative, drought and salt) and to Phelipanche egyptiaca (Egyptian broomrape), an obligate parasitic plant. To this end, tobacco (Nicotiana tabacum) plants were transformed with a bacterial gene (AroG) encode to feedback-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway. Two sets of transgenic plants were obtained: the first had low expression of the AroG protein, a normal phenotype and minor metabolic changes; the second had high accumulation of the AroG protein with normal, or deleterious morphological changes having a dramatic shift in plant metabolism. Metabolic profiling analysis revealed that the leaves of the transgenic plants had increased levels of phenylalanine (up to 43-fold), tyrosine (up to 24-fold) and tryptophan (up to 10-fold) compared to control plants having an empty vector (EV) and wild type (WT) plants. The significant increase in phenylalanine was accompanied by higher levels of metabolites that belong to the phenylpropanoid pathway. AroG plants showed improved tolerance to salt stress but not to oxidative or drought stress. The most significant improved tolerance was to P. aegyptiaca. Unlike WT/EV plants that were heavily infected by the parasite, the transgenic AroG plants strongly inhibited P. aegyptiaca development, and only a few stems of the parasite appeared above the soil. This delayed development of P. aegyptiaca could be the result of higher accumulation of several phenylpropanoids in the transgenic AroG plants and in P. aegyptiaca, that apparently affected its growth. These findings indicate that high levels of AAAs and their related metabolites have the potential of controlling the development of parasitic plants.

Leaf isoprene emission as a trait that mediates the growth-defense tradeoff in the face of climate stress.

Plant isoprene emissions are known to contribute to abiotic stress tolerance, especially during episodes of high temperature and drought, and during cellular oxidative stress. Recent studies have shown that genetic transformations to add or remove isoprene emissions cause a cascade of cellular modifications that include known signaling pathways, and interact to remodel adaptive growth-defense tradeoffs. The most compelling evidence for isoprene signaling is found in the shikimate and phenylpropanoid pathways, which produce salicylic acid, alkaloids, tannins, anthocyanins, flavonols and other flavonoids; all of which have roles in stress tolerance and plant defense. Isoprene also influences key gene expression patterns in the terpenoid biosynthetic pathways, and the jasmonic acid, gibberellic acid and cytokinin signaling networks that have important roles in controlling inducible defense responses and influencing plant growth and development, particularly following defoliation. In this synthesis paper, using past studies of transgenic poplar, tobacco and Arabidopsis, we present the evidence for isoprene acting as a metabolite that coordinates aspects of cellular signaling, resulting in enhanced chemical defense during periods of climate stress, while minimizing costs to growth. This perspective represents a major shift in our thinking away from direct effects of isoprene, for example, by changing membrane properties or quenching ROS, to indirect effects, through changes in gene expression and protein abundances. Recognition of isoprene's role in the growth-defense tradeoff provides new perspectives on evolution of the trait, its contribution to plant adaptation and resilience, and the ecological niches in which it is most effective.

Respiratory chain components are required for peptidoglycan recognition protein-induced thiol depletion and killing in Bacillus subtilis and Escherichia coli

Mammalian peptidoglycan recognition proteins (PGRPs or PGLYRPs) kill bacteria through induction of synergistic oxidative, thiol, and metal stress. Tn-seq screening of Bacillus subtilis transposon insertion library revealed that mutants in the shikimate pathway of chorismate synthesis had high survival following PGLYRP4 treatment. Deletion mutants for these genes had decreased amounts of menaquinone (MK), increased resistance to killing, and attenuated depletion of thiols following PGLYRP4 treatment. These effects were reversed by MK or reproduced by inhibiting MK synthesis. Deletion of cytochrome aa3-600 or NADH dehydrogenase (NDH) genes also increased B. subtilis resistance to PGLYRP4-induced killing and attenuated thiol depletion. PGLYRP4 treatment also inhibited B. subtilis respiration. Similarly in Escherichia coli, deletion of ubiquinone (UQ) synthesis, formate dehydrogenases (FDH), NDH-1, or cytochrome bd-I genes attenuated PGLYRP4-induced thiol depletion. PGLYRP4-induced low level of cytoplasmic membrane depolarization in B. subtilis and E. coli was likely not responsible for thiol depletion. Thus, our results show that the respiratory electron transport chain components, cytochrome aa3-600, MK, and NDH in B. subtilis, and cytochrome bd-I, UQ, FDH-O, and NDH-1 in E. coli, are required for both PGLYRP4-induced killing and thiol depletion and indicate conservation of the PGLYRP4-induced thiol depletion and killing mechanisms in Gram-positive and Gram-negative bacteria.

The entry reaction of the plant shikimate pathway is subjected to highly complex metabolite-mediated regulation.

The plant shikimate pathway directs bulk carbon flow toward biosynthesis of aromatic amino acids (AAAs, i.e. tyrosine, phenylalanine, and tryptophan) and numerous aromatic phytochemicals. The microbial shikimate pathway is feedback inhibited by AAAs at the first enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DHS). However, AAAs generally do not inhibit DHS activities from plant extracts and how plants regulate the shikimate pathway remains elusive. Here, we characterized recombinant Arabidopsis thaliana DHSs (AthDHSs) and found that tyrosine and tryptophan inhibit AthDHS2, but not AthDHS1 or AthDHS3. Mixing AthDHS2 with AthDHS1 or 3 attenuated its inhibition. The AAA and phenylpropanoid pathway intermediates chorismate and caffeate, respectively, strongly inhibited all AthDHSs, while the arogenate intermediate counteracted the AthDHS1 or 3 inhibition by chorismate. AAAs inhibited DHS activity in young seedlings, where AthDHS2 is highly expressed, but not in mature leaves, where AthDHS1 is predominantly expressed. Arabidopsis dhs1 and dhs3 knockout mutants were hypersensitive to tyrosine and tryptophan, respectively, while dhs2 was resistant to tyrosine-mediated growth inhibition. dhs1 and dhs3 also had reduced anthocyanin accumulation under high light stress. These findings reveal the highly complex regulation of the entry reaction of the plant shikimate pathway and lay the foundation for efforts to control the production of AAAs and diverse aromatic natural products in plants.

CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus

Background2-phenylethanol (2-PE) is a rose-scented flavor and fragrance compound that is used in food, beverages, and personal care products. Compatibility with gasoline also makes it a potential biofuel or fuel additive. A biochemical process converting glucose or other fermentable sugars to 2-PE can potentially provide a more sustainable and economical production route than current methods that use chemical synthesis and/or isolation from plant material.ResultsWe work toward this goal by engineering the Shikimate and Ehrlich pathways in the stress-tolerant yeast Kluyveromyces marxianus. First, we develop a multigene integration tool that uses CRISPR-Cas9 induced breaks on the genome as a selection for the one-step integration of an insert that encodes one, two, or three gene expression cassettes. Integration of a 5-kbp insert containing three overexpression cassettes successfully occurs with an efficiency of 51 ± 9% at the ABZ1 locus and was used to create a library of K. marxianus CBS 6556 strains with refactored Shikimate pathway genes. The 33-factorial library includes all combinations of KmARO4, KmARO7, and KmPHA2, each driven by three different promoters that span a wide expression range. Analysis of the refactored pathway library reveals that high expression of the tyrosine-deregulated KmARO4K221L and native KmPHA2, with the medium expression of feedback insensitive KmARO7G141S, results in the highest increase in 2-PE biosynthesis, producing 684 ± 73 mg/L. Ehrlich pathway engineering by overexpression of KmARO10 and disruption of KmEAT1 further increases 2-PE production to 766 ± 6 mg/L. The best strain achieves 1943 ± 63 mg/L 2-PE after 120 h fed-batch operation in shake flask cultures.ConclusionsThe CRISPR-mediated multigene integration system expands the genome-editing toolset for K. marxianus, a promising multi-stress tolerant host for the biosynthesis of 2-PE and other aromatic compounds derived from the Shikimate pathway.

Integrated proteomics and metabolomics analysis of transgenic and gene-stacked maize line seeds

ABSTRACT Unintended effects of genetically modified (GM) crops may pose safety issues. Omics techniques provide researchers with useful tools to assess such unintended effects. Proteomics and metabolomics analyses were performed for three GM maize varieties, 2A-7, CC-2, and 2A-7×CC-2 stacked transgenic maize, and the corresponding non-GM parent Zheng58. Proteomics revealed 120, 271 and 135 maize differentially expressed proteins (DEPs) in the 2A-7/Zheng58, CC-2/Zheng58 and 2A-7×CC-2/Zheng58 comparisons, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that most DEPs participated in metabolic pathways and the biosynthesis of secondary metabolite. Metabolomics revealed 179, 135 and 131 differentially accumulated metabolites (DAMs) in the 2A-7/Zheng58, CC-2/Zheng58 and 2A-7×CC-2/Zheng58 comparisons, respectively. Based on KEGG enrichment analysis, most DAMs are involved in the biosynthesis of secondary metabolite and metabolic pathways. According to integrated proteomics and metabolomics analysis, the introduction of exogenous EPSPS did not affect the expression levels of six other enzymes or the abundance of seven metabolites involved in the shikimic acid pathway in CC-2 and 2A-7×CC-2 seeds. Six co-DEPs annotated by integrated proteomics and metabolomics pathway analysis were further analyzed by qRT-PCR. This study successfully employed integrated proteomic and metabolomic technology to assess unintended changes in maize varieties. The results suggest that GM and gene stacking do not cause significantly unintended effects.

Proteomics analysis reveals that foreign cp4-epsps gene regulates the levels of shikimate and branched pathways in genetically modified soybean line H06-698

ABSTRACT Although genetically modified (GM) glyphosate-resistant soybeans with cp4-epsps gene have been widely planted all over the world, their proteomic characteristics are not very clear. In this study, the soybean seeds of a GM soybean line H06-698 (H) with cp4-epsps gene and its non-transgenic counterpart Mengdou12 (M), which were collected from two experiment fields in two years and used as 4 sample groups, were analyzed with label-free proteomics technique. A total of 1706 proteins were identified quantitatively by label-free quantification, and a total of 293 proteins were detected as common differential abundance proteins (DAPs, FC is not less than 1.5) both in two groups or more. Functional enrichment analysis of common DAPs identified from four groups, shows that most up-regulated proteins were clustered into stress response, carbon and energy metabolism, and genetic information processing. Further documentary analysis shows that 15 proteins play important roles in shikimate pathways, reactive oxygen species (ROS) and stress response. These results indicated that the change of protein abundance in different samples were affected by various factors, but except shikimate and branched pathways related proteins, only ROS and stress-related proteins were found to be stably regulated by cp4-epsps gene, and no unexpected and safety-related proteins such as antinutritional factors, allergenic proteins, and toxic proteins were found as DAPs. The influence of foreign genes in genetically modified plants is worthy of attention and this work provides new clues for exploring the regulated proteins and pathways in GM plants.

Use of Shotgun Metagenomics and Metabolomics to Evaluate the Impact of Glyphosate or Roundup MON 52276 on the Gut Microbiota and Serum Metabolome of Sprague-Dawley Rats.

Background:There is intense debate on whether glyphosate can inhibit the shikimate pathway of gastrointestinal microorganisms, with potential health implications.Objectives:We tested whether glyphosate or its representative EU herbicide formulation Roundup MON 52276 affects the rat gut microbiome.Methods:We combined cecal microbiome shotgun metagenomics with serum and cecum metabolomics to assess the effects of glyphosate [0.5, 50, ] or MON 52276 at the same glyphosate-equivalent doses, in a 90-d toxicity test in rats.Results:Glyphosate and MON 52276 treatment resulted in ceca accumulation of shikimic acid and 3-dehydroshikimic acid, suggesting inhibition of 5-enolpyruvylshikimate-3-phosphate synthase of the shikimate pathway in the gut microbiome. Cysteinylglycine, , and valylglycine levels were elevated in the cecal microbiome following glyphosate and MON 52276 treatments. Altered cecum metabolites were not differentially expressed in serum, suggesting that the glyphosate and MON 52276 impact on gut microbial metabolism had limited consequences on physiological biochemistry. Serum metabolites differentially expressed with glyphosate treatment were associated with nicotinamide, branched-chain amino acid, methionine, cysteine, and taurine metabolism, indicative of a response to oxidative stress. MON 52276 had similar, but more pronounced, effects than glyphosate on the serum metabolome. Shotgun metagenomics of the cecum showed that treatment with glyphosate and MON 52276 resulted in higher levels of Eggerthella spp., Shinella zoogleoides, Acinetobacter johnsonii, and Akkermansia muciniphila. Shinella zoogleoides was higher only with MON 52276 exposure. In vitro culture assays with Lacticaseibacillus rhamnosus strains showed that Roundup GT plus inhibited growth at concentrations at which MON 52276 and glyphosate had no effect.Discussion:Our study highlights the power of multi-omics approaches to investigate the toxic effects of pesticides. Multi-omics revealed that glyphosate and MON 52276 inhibited the shikimate pathway in the rat gut microbiome. Our findings could be used to develop biomarkers for epidemiological studies aimed at evaluating the effects of glyphosate herbicides on humans. https://doi.org/10.1289/EHP6990

In vitro characterization of 3-chloro-4-hydroxybenzoic acid building block formation in ambigol biosynthesis.

The cyanobacterium Fischerella ambigua is a natural producer of polychlorinated aromatic compounds, the ambigols A-E. The biosynthetic gene cluster (BGC) of these highly halogenated triphenyls has been recently identified by heterologous expression. It consists of 10 genes named ab1-10. Two of the encoded enzymes, i.e. Ab2 and Ab3, were identified by in vitro and in vivo assays as cytochrome P450 enzymes responsible for biaryl and biaryl ether formation. The key substrate for these P450 enzymes is 2,4-dichlorophenol, which in turn is derived from the precursor 3-chloro-4-hydroxybenzoic acid. Here, the biosynthetic steps leading towards 3-chloro-4-hydroxybenzoic acid were investigated by in vitro assays. Ab7, an isoenzyme of a 3-deoxy-7-phosphoheptulonate (DAHP) synthase, is involved in chorismate biosynthesis by the shikimate pathway. Chorismate in turn is further converted by a dedicated chorismate lyase (Ab5) yielding 4-hydroxybenzoic acid (4-HBA). The stand alone adenylation domain Ab6 is necessary to activate 4-HBA, which is subsequently tethered to the acyl carrier protein (ACP) Ab8. The Ab8 bound substrate is chlorinated by Ab10 in meta position yielding 3-Cl-4-HBA, which is then transfered by the condensation (C) domain to the peptidyl carrier protein and released by the thioesterase (TE) domain of Ab9. The released product is then expected to be the dedicated substrate of the halogenase Ab1 producing the monomeric ambigol building block 2,4-dichlorophenol.

A bioactive molecule made by unusual salvage of radical SAM enzyme byproduct 5-deoxyadenosine blurs the boundary of primary and secondary metabolism

5-Deoxyadenosine (5dAdo) is the byproduct of many radical S-adenosyl-l-methionine enzyme reactions in all domains of life. 5dAdo is also an inhibitor of the radical S-adenosyl-l-methionine enzymes themselves, making it necessary for cells to construct pathways to recycle or dispose of this toxic metabolite. However, the specific pathways involved have long remained unexplored. Recent research demonstrated a growth advantage in certain organisms by using 5dAdo or intermediates as a sole carbon source and elucidated the corresponding salvage pathway. We now provide evidence using supernatant analysis by GC–MS for another 5dAdo recycling route. Specifically, in the unicellular cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus), the activity of promiscuous enzymes leads to the synthesis and excretion first of 5-deoxyribose and subsequently of 7-deoxysedoheptulose. 7-Deoxysedoheptulose is an unusual deoxy-sugar, which acts as an antimetabolite of the shikimate pathway, thereby exhibiting antimicrobial and herbicidal activity. This strategy enables organisms with small genomes and lacking canonical gene clusters for the synthesis of secondary metabolites, like S. elongatus, to produce antimicrobial compounds from primary metabolism and enzymatic promiscuity. Our findings challenge the view of bioactive molecules as sole products of secondary metabolite gene clusters and expand the range of compounds that microorganisms can deploy to compete for their ecological niche.

Yarrowia lipolytica chassis strains engineered to produce aromatic amino acids via the shikimate pathway.

Summary Yarrowia lipolytica is widely used as a microbial producer of lipids and lipid derivatives. Here, we exploited this yeast’s potential to generate aromatic amino acids by developing chassis strains optimized for the production of phenylalanine, tyrosine and tryptophan. We engineered the shikimate pathway to overexpress a combination of Y. lipolytica and heterologous feedback‐insensitive enzyme variants. Our best chassis strain displayed high levels of de novo Ehrlich metabolite production (up to 0.14 g l−1 in minimal growth medium), which represented a 93‐fold increase compared to the wild‐type strain (0.0015 g l−1). Production was further boosted to 0.48 g l−1 when glycerol, a low‐cost carbon source, was used, concomitantly to high secretion of phenylalanine precursor (1 g l−1). Among these metabolites, 2‐phenylethanol is of particular interest due to its rose‐like flavour. We also established a production pathway for generating protodeoxyviolaceinic acid, a dye derived from tryptophan, in a chassis strain optimized for chorismate, the precursor of tryptophan.We have thus demonstrated that Y. lipolytica can serve as a platform for the sustainable de novo bio‐production of high‐value aromatic compounds, and we have greatly improved our understanding of the potential feedback‐based regulation of the shikimate pathway in this yeast.

Dehydroquinate dehydratase/shikimate dehydrogenases involved in gallate biosynthesis of the aluminum-tolerant tree species Eucalyptus camaldulensis

Main conclusionEucalyptus camaldulensis EcDQD/SDH2 and 3 combine gallate formation, dehydroquinate dehydratase, and shikimate dehydrogenase activities. They are candidates for providing the essential gallate for the biosynthesis of the aluminum-detoxifying metabolite oenothein B.The tree species Eucalyptus camaldulensis shows exceptionally high tolerance against aluminum, a widespread toxic metal in acidic soils. In the roots of E. camaldulensis, aluminum is detoxified via the complexation with oenothein B, a hydrolyzable tannin. In our approach to elucidate the biosynthesis of oenothein B, we here report on the identification of E. camaldulensis enzymes that catalyze the formation of gallate, which is the phenolic constituent of hydrolyzable tannins. By systematical screening of E. camaldulensis dehydroquinate dehydratase/shikimate dehydrogenases (EcDQD/SDHs), we found two enzymes, EcDQD/SDH2 and 3, catalyzing the NADP+-dependent oxidation of 3-dehydroshikimate to produce gallate. Based on extensive in vitro assays using recombinant EcDQD/SDH2 and 3 enzymes, we present for the first time a detailed characterization of the enzymatic gallate formation activity, including the cofactor preferences, pH optima, and kinetic constants. Sequence analyses and structure modeling suggest the gallate formation activity of EcDQD/SDHs is based on the reorientation of 3-dehydroshikimate in the catalytic center, which facilitates the proton abstraction from the C5 position. Additionally, EcDQD/SDH2 and 3 maintain DQD and SDH activities, resulting in a 3-dehydroshikimate supply for gallate formation. In E. camaldulensis, EcDQD/SDH2 and 3 are co-expressed with UGT84A25a/b and UGT84A26a/b involved in hydrolyzable tannin biosynthesis. We further identified EcDQD/SDH1 as a “classical” bifunctional plant shikimate pathway enzyme and EcDQD/SDH4a/b as functional quinate dehydrogenases of the NAD+/NADH-dependent clade. Our data indicate that in E. camaldulensis the enzymes EcDQD/SDH2 and 3 provide the essential gallate for the biosynthesis of the aluminum-detoxifying metabolite oenothein B.

Chorismate synthase mediates cerebral malaria pathogenesis by eliciting salicylic acid-dependent autophagy response in parasite.

ABSTRACTCerebral malaria caused by Plasmodium falciparum is the severest form of the disease resulting in the morbidity of a huge number of people worldwide. Development of effective curatives is essential in order to overcome the fatality of cerebral malaria. Earlier studies have shown the presence of salicylic acid (SA) in malaria parasite P. falciparum, which plays a critical role in the manifestation of cerebral malaria. Further, the application of SA for the treatment of acute symptoms in cerebral malaria increases the activity of iNOS leading to severe inflammation-mediated death, also called as Reye's syndrome. Therefore, modulation of the level of SA might be a novel approach to neutralize the symptoms of cerebral malaria. The probable source of parasite SA is the shikimate pathway, which produces chorismate, a precursor to aromatic amino acids and other secondary metabolites like SA in the parasite. In this work, we performed the immunological, pathological and biochemical studies in mice infected with chorismate synthase knocked-out Plasmodium berghei ANKA, which does not produce SA. Fewer cerebral outcomes were observed as compared to the mice infected with wild-type parasite. The possible mechanism behind this protective effect might be the hindrance of SA-mediated induction of autophagy in the parasite, which helps in its survival in the stressed condition of brain microvasculature during cerebral malaria. The absence of SA leading to reduced parasite load along with the reduced pathological symptoms contributes to less fatality outcome by cerebral malaria.

Precursor-directed Biosynthesis in Tabernaemontana catharinensis as a New Avenue for Alzheimer's Disease-modifying Agents.

Plants produce a high diversity of metabolites that can act as regulators of cholinergic dysfunction. Among plants, the potential of species of the genus Tabernaemontana to treat neurological disorders has been linked to iboga-type alkaloids that are biosynthesized by those species. In this context, precursor-directed biosynthesis approaches were carried out using T. catharinensis plantlets to achieve new-to-nature molecules as promising agents against Alzheimer's disease. Aerial parts of T. catharinensis, cultured in vitro, produced 7 unnatural alkaloids (5-fluoro-ibogamine, 5-fluoro-voachalotine, 5-fluoro-12-methoxy-Nb-methyl-voachalotine, 5-fluoro-isovoacangine, 5-fluoro-catharanthine, 5-fluoro-19-(S)-hydroxy-ibogamine, and 5-fluoro-coronaridine), while root extracts showed the presence of the same unnatural iboga-type alkaloids and 2 additional ones: 5-fluoro-voafinine and 5-fluoro-affinisine. Moreover, molecular docking approaches were carried out to evaluate the potential inhibition activity of T. catharinensis' natural and unnatural alkaloids against AChE and BChE enzymes. Fluorinated iboga alkaloids (5-fluoro-catharanthine, 5-fluoro-voachalotine, 5-fluoro-affinisine, 5-fluoro-isovoacangine, 5-fluoro-corinaridine) were more active than natural ones and controls against AchE, while 5-fluoro-19-(S)-hydroxy-ibogamine, 5-fluoro-catharanthine, 5-fluoro-isovoacangine, and 5-fluoro-corinaridine showed better activity than natural ones and controls against BChE. Our findings showed that precursor-directed biosynthesis strategies generated "new-to-nature" alkaloids that are promising Alzheimer's disease drug candidates. Furthermore, the isotopic experiments also allowed us to elucidate the initial steps of the biosynthetic pathway for iboga-type alkaloids, which are derived from the MEP and shikimate pathways.