Eremoxylarins D-J, Antibacterial Eremophilane Sesquiterpenes Discovered from an Endolichenic Strain of Xylaria hypoxylon.

An endolichenic strain of the Ascomycetaceous Xylaria hypoxylon, cultivated alone or in coculture with another endolichenic fungus Dendrothyrium variisporum, produced seven new bioactive eremophilane sesquiterpenes eremoxylarins D-J (1-7). The isolated compounds disclosed a high similarity with the eremophilane core of the bioactive integric acid, and structures were elucidated by 1D and 2D NMR spectra and electronic circular dichroism (ECD) analyses. Eremoxylarins D, F, G, and I showed a selective activity against Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus with minimum inhibitory concentration (MIC) values between 0.39 and 12.5 µg/mL. Eremoxylarin I, the most antibacterial active sesquiterpene, was also active against HCoV-229E at a concentration nontoxic to the hepatoma Huh-7 cell line with an 50% inhibitory concentration (IC50) of 18.1 µM and a 50% cytotoxic concentration (CC50) of 46.6 µM.

Volatile Organic Compounds from a Lichen-Associated Bacterium, Paenibacillus etheri, Interact with Plant-Parasitic Cyst Nematodes.

Healthy food is one of the major challenges to develop in this century. Plant-parasitic nematodes cause significant damage to many crops worldwide and till now, the use of chemical nematicides is the main means to control their populations. These chemical products must be replaced by more environmental-friendly control methods. Biocontrol methods seem to be one promising option, and the number of biopesticides derived from living organisms has increased in the last decades. To develop new plant protection products, we have decided to combine our skills in natural products chemistry and nematology and to focus on the lichen microecosystem as underexploited ecological niches of microorganisms. We present herein the potential of lichen-associated bacterial suspensions from Paenibacillus etheri as nematicides against the beet cyst nematode Heterodera schachtii and the potato cyst nematode Globodera pallida, in particular the effects of volatile organic compounds (VOCs) produced by the bacteria. A solid phase micro-extraction method associated to gas chromatography-mass spectrometry analysis of 14 day cultures was used to analyze these VOCs in order to identify the main produced compounds (isoamyl acetate and 2-phenylethyl acetate) and to evaluate them on the nematodes.

Rhizocarpon geographicum Lichen Discloses a Highly Diversified Microbiota Carrying Antibiotic Resistance and Persistent Organic Pollutant Tolerance.

As rock inhabitants, lichens are exposed to extreme and fluctuating abiotic conditions associated with poor sources of nutriments. These extreme conditions confer to lichens the unique ability to develop protective mechanisms. Consequently, lichen-associated microbes disclose highly versatile lifestyles and ecological plasticity, enabling them to withstand extreme environments. Because of their ability to grow in poor and extreme habitats, bacteria associated with lichens can tolerate a wide range of pollutants, and they are known to produce antimicrobial compounds. In addition, lichen-associated bacteria have been described to harbor ecological functions crucial for the evolution of the lichen holobiont. Nevertheless, the ecological features of lichen-associated microbes are still underestimated. To explore the untapped ecological diversity of lichen-associated bacteria, we adopted a novel culturomic approach on the crustose lichen Rhizocarpon geographicum. We sampled R. geographicum in French habitats exposed to oil spills, and we combined nine culturing methods with 16S rRNA sequencing to capture the greatest bacterial diversity. A deep functional analysis of the lichen-associated bacterial collection showed the presence of a set of bacterial strains resistant to a wide range of antibiotics and displaying tolerance to Persistent Organic Pollutants (POPs). Our study is a starting point to explore the ecological features of the lichen microbiota.

Microbial community associated with the crustose lichen Rhizocarpon geographicum L. (DC.) living on oceanic seashore: A large source of diversity revealed by using multiple isolation methods.

Recently, the study of the interactions within a microcosm between hosts and their associated microbial communities drew an unprecedented interest arising from the holobiont concept. Lichens, a symbiotic association between a fungus and an alga, are redefined as complex ecosystems considering the tremendous array of associated microorganisms that satisfy this concept. The present study focuses on the diversity of the microbiota associated with the seashore located lichen Rhizocarpon geographicum, recovered by different culture-dependent methods. Samples harvested from two sites allowed the isolation and the molecular identification of 68 fungal isolates distributed in 43 phylogenetic groups, 15 bacterial isolates distributed in five taxonomic groups and three microalgae belonging to two species. Moreover, for 12 fungal isolates belonging to 10 different taxa, the genus was not described in GenBank. These fungal species have never been sequenced or described and therefore non-studied. All these findings highlight the novel and high diversity of the microflora associated with R. geographicum. While many species disappear every day, this work suggests that coastal and wild environments still contain an unrevealed variety to offer and that lichens constitute a great reservoir of new microbial taxa which can be recovered by multiplying the culture-dependent techniques.

Glycoside Hydrolases and Glycosyltransferases from Hyperthermophilic Archaea: Insights on Their Characteristics and Applications in Biotechnology.

Hyperthermophilic Archaea colonizing unnatural habitats of extremes conditions such as volcanoes and deep-sea hydrothermal vents represent an unmeasurable bioresource for enzymes used in various industrial applications. Their enzymes show distinct structural and functional properties and are resistant to extreme conditions of temperature and pressure where their mesophilic homologs fail. In this review, we will outline carbohydrate-active enzymes (CAZymes) from hyperthermophilic Archaea with specific focus on the two largest families, glycoside hydrolases (GHs) and glycosyltransferases (GTs). We will present the latest advances on these enzymes particularly in the light of novel accumulating data from genomics and metagenomics sequencing technologies. We will discuss the contribution of these enzymes from hyperthermophilic Archaea to industrial applications and put the emphasis on newly identifed enzymes. We will highlight their common biochemical and distinct features. Finally, we will overview the areas that remain to be explored to identify novel promising hyperthermozymes.

Modulation of the Activity and Regioselectivity of a Glycosidase: Development of a Convenient Tool for the Synthesis of Specific Disaccharides.

The synthesis of disaccharides, particularly those containing hexofuranoside rings, requires a large number of steps by classical chemical means. The use of glycosidases can be an alternative to limit the number of steps, as they catalyze the formation of controlled glycosidic bonds starting from simple and easy to access building blocks; the main drawbacks are the yields, due to the balance between the hydrolysis and transglycosylation of these enzymes, and the enzyme-dependent regioselectivity. To improve the yield of the synthesis of ß-d-galactofuranosyl-(1→X)-d-mannopyranosides catalyzed by an arabinofuranosidase, in this study we developed a strategy to mutate, then screen the catalyst, followed by a tailored molecular modeling methodology to rationalize the effects of the identified mutations. Two mutants with a 2.3 to 3.8-fold increase in transglycosylation yield were obtained, and in addition their accumulated regioisomer kinetic profiles were very different from the wild-type enzyme. Those differences were studied in silico by docking and molecular dynamics, and the methodology revealed a good predictive quality in regards with the regioisomer profiles, which is in good agreement with the experimental transglycosylation kinetics. So, by engineering CtAraf51, new biocatalysts were enabled to obtain the attractive central motif from the Leishmania lipophosphoglycan core with a higher yield and regioselectivity.

Lipase-Catalyzed Ring-Opening Polymerization of Benzyl Malolactonate: An Unusual Mechanism?

The use of safe natural catalyst such as enzymes for ring opening polymerization (ROP) of ß-substituted ß-lactones such as benzyl malolactonate (MLABe) is an important objective considering the biomedical applications of the resulting (co)polymers. However, the preparation of well-defined polymeric materials using such systems requires an understanding of enzyme-substrate interactions. In this context, we investigated the mechanism of lipase-catalyzed ROP of MLABe, because it appears that it is probably not the same as the one widely described for other lactones such ε-caprolactone, propiolactone. and lactide. Enzymatic-catalyzed ROPs of MLABe in the presence of the lipase/acyltransferase CpLip2 and its serine knockout (serine KO) mutant (CpLip2_180A) have led to poly(benzyl malate) (PMLABe) terminated by a monobenzyl fumarate group with monomer conversion higher than 70% and weight-average molar mass of about 3600 g/mol (D = 1.42). On the other hand, only less than 7% of MLABe conversion and no polymer formation were observed when the polymerization reaction was conducted in the presence of inactivated CpLip2 (heated at 100 °C). Moreover, the ROP of MLABe in the presence of imidazole, a synthetic mimic of the catalytic histidine, led to a PMLABe terminated by a monobenzyl fumarate group. On the contrary, neither the enzymatic-catalyzed ROP of benzyl dimethylmalolactonate (diMeMLABe), a MLABe with two methyl groups instead of the two "acidic" protons on the lactone's ring, in the presence of CpLip2 and CpLip2_180A nor its chemical ROP in the presence of imidazole were successful. Together, all these results suggested that the lipase-catalyzed polymerization of malolactonates occurred through the abstraction of one of the two "acidic" protons of the lactone's ring by the histidine of the catalytic triad leading to the corresponding monobenzyl fumarate responsible for the polymerization of the remaining monomer. Finally, molecular modeling of the positioning of the monomer into the catalytic site of the CpLip2 and DFT quantum-chemical calculations highlighted an interaction of (R)- and (S)-MLABe with the catalytic histidine of the enzyme preferentially to serine, in the form of a strong hydrogen bond with one of the "acidic" protons of MLABe, thus, supporting the important role of the catalytic histidine in the polymerization of such cyclic lactones.

Improvement of the versatility of an arabinofuranosidase against galactofuranose for the synthesis of galactofuranoconjugates.

Galactofuranoconjugates are rare compounds with interesting biological properties. Their syntheses by traditional approaches are however tedious. Glycosidases are nowadays often used to simplify such syntheses but the use of galactofuranosidase has not been described yet for the synthesis of galactofuranoconjugates. Interestingly CtAraf51, an α-l-arabinofuranosidase from Ruminiclostridium thermocellum, is able to use aryl- or alkyl-ß-d-galactofuranosides as the substrate but with very low efficiency. To allow its use as a synthesis tool, we decided to improve the efficiency of this enzyme toward these non-natural substrates. First, we identified three residues that can contribute to unfavorable interactions with the p-nitrophenyl-ß-d-galactofuranoside. After mutagenesis, two mutants have shown a catalytic efficiency four- and threefold higher than that of the wild type, respectively. These two mutants were then evaluated in the transglycosylation reaction using ethanol as a model acceptor substrate. Under these conditions one mutant was much more efficient: 50% conversion was reached ten times faster than with the WT. Finally both mutants were converted into thioglycoligases: in the thioligation reaction, the reaction was two times faster than with the E173A single mutant, and in the acylation reaction a fourfold increase in the initial velocity was found. The synthetic potential of the resulting mutants to synthesize various O-, S- and acyl galactofuranoconjugates was further evaluated and yields up to 82% were obtained for the synthesis of ethyl- or thiophenyl galactofuranosides and methoxybenzoic galactofuranose.

Diversion of a thioglycoligase for the synthesis of 1-O-acyl arabinofuranoses.

An arabinofuranosylhydrolase from the GH51 family was transformed into an acyl transferase by mutation of the catalytic acid/base amino acid. The resulting enzyme was able to transfer carboxylic acid onto the anomeric position of arabinose with complete chemo- and stereoselectivity. A wide range of acyl α-l-arabinofuranoses was obtained with yields ranging from 25 to 83%. Using this method, ibuprofen and N-Boc phenylalanine were successfully transformed into their corresponding acyl conjugates, expanding the scope of the reaction to drugs and amino acids.

Cell Uptake and Biocompatibility of Nanoparticles Prepared from Poly(benzyl malate) (Co)polymers Obtained through Chemical and Enzymatic Polymerization in Human HepaRG Cells and Primary Macrophages.

The design of drug-loaded nanoparticles (NPs) appears to be a suitable strategy for the prolonged plasma concentration of therapeutic payloads, higher bioavailability, and the reduction of side effects compared with classical chemotherapies. In most cases, NPs are prepared from (co)polymers obtained through chemical polymerization. However, procedures have been developed to synthesize some polymers via enzymatic polymerization in the absence of chemical initiators. The aim of this work was to compare the acute in vitro cytotoxicities and cell uptake of NPs prepared from poly(benzyl malate) (PMLABe) synthesized by chemical and enzymatic polymerization. Herein, we report the synthesis and characterization of eight PMLABe-based polymers. Corresponding NPs were produced, their cytotoxicity was studied in hepatoma HepaRG cells, and their uptake by primary macrophages and HepaRG cells was measured. In vitro cell viability evidenced a mild toxicity of the NPs only at high concentrations/densities of NPs in culture media. These data did not evidence a higher biocompatibility of the NPs prepared from enzymatic polymerization, and further demonstrated that chemical polymerization and the nanoprecipitation procedure led to biocompatible PMLABe-based NPs. In contrast, NPs produced from enzymatically synthesized polymers were more efficiently internalized than NPs produced from chemically synthesized polymers. The efficient uptake, combined with low cytotoxicity, indicate that PMLABe-based NPs are suitable nanovectors for drug delivery, deserving further evaluation in vivo to target either hepatocytes or resident liver macrophages.

Leishmania cell wall as a potent target for antiparasitic drugs. A focus on the glycoconjugates.

Although leishmaniasis has been studied for over a century, the fight against cutaneous, mucocutaneous and visceral forms of the disease remains a hot topic. This review refers to the parasitic cell wall and more particularly to the constitutive glycoconjugates. The structures of the main glycolipids and glycoproteins, which are species-dependent, are described. The focus is on the disturbance of the lipid membrane by existing drugs and possible new ones, in order to develop future therapeutic agents.

An abscisic acid-independent oxylipin pathway controls stomatal closure and immune defense in Arabidopsis.

Plant stomata function in innate immunity against bacterial invasion and abscisic acid (ABA) has been suggested to regulate this process. Using genetic, biochemical, and pharmacological approaches, we demonstrate that (i) the Arabidopsis thaliana nine-specific-lipoxygenase encoding gene, LOX1, which is expressed in guard cells, is required to trigger stomatal closure in response to both bacteria and the pathogen-associated molecular pattern flagellin peptide flg22; (ii) LOX1 participates in stomatal defense; (iii) polyunsaturated fatty acids, the LOX substrates, trigger stomatal closure; (iv) the LOX products, fatty acid hydroperoxides, or reactive electrophile oxylipins induce stomatal closure; and (v) the flg22-mediated stomatal closure is conveyed by both LOX1 and the mitogen-activated protein kinases MPK3 and MPK6 and involves salicylic acid whereas the ABA-induced process depends on the protein kinases OST1, MPK9, or MPK12. Finally, we show that the oxylipin and the ABA pathways converge at the level of the anion channel SLAC1 to regulate stomatal closure. Collectively, our results demonstrate that early biotic signaling in guard cells is an ABA-independent process revealing a novel function of LOX1-dependent stomatal pathway in plant immunity.

Treponema denticola PurE Is a bacterial AIR carboxylase.

De novo purine biosynthesis proceeds by two divergent paths. In bacteria, yeasts, and plants, 5-aminoimidazole ribonucleotide (AIR) is converted to 4-carboxy-AIR (CAIR) by two enzymes: N(5)-carboxy-AIR (N(5)-CAIR) synthetase (PurK) and N(5)-CAIR mutase (class I PurE). In animals, the conversion of AIR to CAIR requires a single enzyme, AIR carboxylase (class II PurE). The CAIR carboxylate derives from bicarbonate or CO(2), respectively. Class I PurE is a promising antimicrobial target. Class I and class II PurEs are mechanistically related but bind different substrates. The spirochete dental pathogen Treponema denticola lacks a purK gene and contains a class II purE gene, the hallmarks of CO(2)-dependent CAIR synthesis. We demonstrate that T. denticola PurE (TdPurE) is AIR carboxylase, the first example of a prokaryotic class II PurE. Steady-state and pre-steady-state experiments show that TdPurE binds AIR and CO(2) but not N(5)-CAIR. Crystal structures of TdPurE alone and in complex with AIR show a conformational change in the key active site His40 residue that is not observed for class I PurEs. A contact between the AIR phosphate and a differentially conserved residue (TdPurE Lys41) enforces different AIR conformations in each PurE class. As a consequence, the TdPurE·AIR complex contains a portal that appears to allow the CO(2) substrate to enter the active site. In the human pathogen T. denticola, purine biosynthesis should depend on available CO(2) levels. Because spirochetes lack carbonic anhydrase, the corresponding reduction in bicarbonate demand may confer a selective advantage.

The rutin catabolic pathway with special emphasis on quercetinase.

The aim of this review is to give a general account on the oxidative microbial degradation of flavonols. Since now 50 years, various research groups have deciphered the way microorganisms aerobically deal with this important class of flavonoids. Flavonols such as rutin and quercetin are abundantly found in vegetal tissues and exudates, and it was thus patent that various microorganisms will bear the enzymatic machinery necessary to cope with these vegetal secondary metabolites. After initial studies focussed on the general metabolic capacity of various microorganisms towards flavonols, the so called rutin catabolic pathway was rapidly established in moulds. Enzymes of the path as well as substrates and products were known at the beginning of the seventies. Then during 30 years, only sporadic studies were focused on this pathway, before a new burst of interest at the beginning of the new century arose with structural, genomic and theoretical studies mainly conducted towards quercetinase. This is the goal of this work to relate this 50 years journey at the crossroads of microbiology, biochemistry, genetic and chemistry. Some mention of the potential usefulness of the enzymes of the path as well as micro-organisms bearing the whole rutin catabolic pathway is also discussed.

Biochemical and molecular characterization of a quercetinase from Penicillium olsonii.

Quercetinase (quercetin 2,3-dioxygenase, EC 1.13.11.24) is produced by various filamentous fungi when grown on rutin as the sole carbon and energy source. From a rutin based liquid culture of Penicillium olsonii, we purified a quercetinase with a specific activity of 175U mg(-1). The enzyme is a monomeric glycoprotein of approximately 55 kDa, containing 0.9+/-0.1 copper atoms per protein. Its substrate specificity is restricted to the flavonol family of flavonoids. It is completely inhibited by diethyldithiocarbamate at a concentration of 100 nM and 1H-2-benzyl-3-hydroxy-4-oxoquinolin is a competitive inhibitor with a K(I) of 4 microM. The cDNA poquer1 was cloned and sequenced. It encodes a 365 amino acids long enzyme with a strong sequence identity with the Aspergillus japonicus quercetinase (Q7SIC2). Like the enzyme from A. japonicus, only one of the two cupin domains of the Penicillium olsonii quercetinase is able to bind a metal atom.

Evaluation of phenolics and sugars as inducers of quercetinase activity in Penicillium olsonii.

Quercetinase is produced by various filamentous fungi when grown on rutin as sole carbon and energy source. We investigated on the effect of 10 phenolics and two sugars, structurally related to substrates and products of the rutin catabolic pathway, on the induction of a quercetinase activity in Penicillium olsonii. Neither the sugars (glucose and rhamnose, two constituents of rutin), nor phenolics such as protocatechuic acid, salicylic acid, 4-hydroxy-benzoic acid and phloroglucinol were inducers. Rutin (maximum activity 150 nmol/min/mL after 5 days), quercetin (70 nmol/min/mL, 3 days), phloroglucinol carboxylic acid (60 nmol/min/mL, 3 days), 2-protocatechuoylphloroglucinolcarboxylic acid (50 nmol/min/mL, 5 days), 2,6-dihydroxy-carboxylic acid (90 nmol/min/mL, 7 days) and 2,4-dihydroxy-carboxylic acid (30 nmol/min/mL, 7 days) were demonstrated to be quercetinase inducers. We propose that rutin, quercetin and 2-protocatechuoyl-phloroglucinol carboxylic acid, the product of the reaction catalysed by quercetinase, act as inducers after their catabolic transformation in phloroglucinol carboxylic acid.