Vincent Phalip

Diversity of the exoproteome of Fusarium graminearum grown on plant cell wall

The exoproteome of the fungus Fusarium graminearum grown on glucose and on hop (Humulus lupulus, L.) cell wall has been investigated. The culture medium was found to contain a higher quantity of proteins and the proteins are more diverse when the fungus is grown on cell wall. Using both 1D and 2D electrophoresis followed by mass spectrometry analysis and protein identification based on similarity searches, 84 unique proteins were identified in the cell wall-grown fungal exoproteome. Many are putatively implicated in carbohydrate metabolism, mainly in cell wall polysaccharide degradation. The predicted carbohydrate-active enzymes fell into 24 different enzymes classes, and up to eight different proteins within a same class are secreted. This indicates that fungal metabolism becomes oriented towards synthesis and secretion of a whole arsenal of enzymes able to digest almost the complete plant cell wall. Cellobiohydrolase is one of the only four proteins found both after growth on glucose and on plant cell wall and we propose that this enzyme could act as a sensor of the extracellular environment. Extensive knowledge of this very diverse F. graminearum exoproteome is an important step towards the full understanding of Fusarium/plants interactions.

Molecular basis of arabinobio-hydrolase activity in phytopathogenic fungi: crystal structure and catalytic mechanism of Fusarium graminearum GH93 exo-alpha-L-arabinanase.

The phytopathogenic fungus Fusarium graminearum secretes a very diverse pool of glycoside hydrolases (GHs) aimed at degrading plant cell walls. α-l-Arabinanases are essential GHs participating in the complete hydrolysis of hemicellulose, a natural resource for various industrial processes, such as bioethanol or pharmaceuticals production. Arb93A, the exo-1,5-α-l-arabinanase of F. graminearum encoded by the gene fg03054.1, belongs to the GH93 family, for which no structural data exists. The enzyme is highly active (1065 units/mg) and displays a strict substrate specificity for linear α-1,5-l-arabinan. Biochemical assays and NMR experiments demonstrated that the enzyme releases α-1,5-l-arabinobiose from the nonreducing end of the polysaccharide. We determined the crystal structure of the native enzyme and its complex with α-1,5-l-arabinobiose, a degradation product of α-Me-1,5-l-arabinotetraose, at 1.85 and 2.05Å resolution, respectively. Arb93A is a monomeric enzyme, which presents the six-bladed β-propeller fold characteristic of sialidases of clan GHE. The configuration of the bound arabinobiose is consistent with the retaining mechanism proposed for the GH93 family. Catalytic residues were proposed from the structural analysis, and site-directed mutagenesis was used to validate their role. They are significantly different from those observed for GHE sialidases.

Product patterns of a feruloyl esterase from Aspergillus nidulans on large feruloyl-arabino-xylo-oligosaccharides from wheat bran.

A purified feruloyl esterase (EC 3.1.1.73) from Aspergillus nidulans produced in Pichia pastoris was used to study the de-esterification of large feruloyl oligosaccharides consisting of 4 to 20 pentose residues and (xylose plus arabinose) and one ferulic acid residue. The feruloyl oligosaccharides were prepared from total oligosaccharidic hydrolysates from wheat bran treated with a purified endoxylanase from Thermobacillus xylanilyticus. The feruloyl esterase showed similar specific activity but an affinity about 3.5-fold higher towards feruloyl oligosaccharides than towards methyl ferulate. Mass spectrometry analysis of the products after long-term enzymatic hydrolyses showed that the esterase was able to hydrolyze the largest feruloyl oligosaccharides and therefore could act alone on feruloyled xylans. Consequently, the feruloyl esterase from A. nidulans could be useful for the enzymatic deconstruction of xylans in plant cell walls.

Amylases without known homologues discovered in an acid mine drainage: significance and impact

Acid Mine Drainages (AMDs) are extreme environments characterized by acidic and oligotrophic conditions and by metal contaminations. A function-based screening of an AMD-derived metagenomic library led to the discovery and partial characterization of two non-homologous endo-acting amylases sharing no sequence similarity with any known amylase nor glycosidase. None carried known amylolytic domains, nor could be assigned to any GH-family. One amylase displayed no similarity with any known protein, whereas the second one was similar to TraC proteins involved in the bacterial type IV secretion system. According to the scarce similarities with known proteins, 3D-structure modelling using I-TASSER was unsuccessful. This study underlined the utility of a function-driven metagenomic approach to obtain a clearer image of the bacterial community enzymatic landscape. More generally, this work points out that screening for microorganisms or biomolecules in a priori incongruous environments could provide unconventional and new exciting ways for bioprospecting.

Fungal Fermentation of Lignocellulosic Biomass for Itaconic and Fumaric Acid Production

The production of high-value chemicals from natural resources as an alternative for petroleum-based products is currently expanding in parallel with biorefinery. The use of lignocellulosic biomass as raw material is promising to achieve economic and environmental sustainability. Filamentous fungi, particularly Aspergillus species, are already used industrially to produce organic acid as well as many enzymes. The production of lignocellulose-degrading enzymes opens the possibility for direct fungal fermentation towards organic acids such as itaconic acid (IA) and fumaric acid (FA). These acids have wide-range applications and potentially addressable markets as platform chemicals. However, current technologies for the production of these compounds are mostly based on submerged fermentation. This work showed the capacity of two Aspergillus species (A. terreus and A. oryzae) to yield both acids by solid-state fermentation and simultaneous saccharification and fermentation. FA was optimally produced at by A. oryzae in simultaneous saccharification and fermentation (0.54 mg/g wheat bran). The yield of 0.11 mg IA/g biomass by A. oryzae is the highest reported in the literature for simultaneous solid-state fermentation without sugar supplements.

Genome‐wide transcriptional responses of Fusarium graminearum to plant cell wall substrates

We report a genome-wide transcriptomic study of Fusarium graminearum grown on four different substrates based on plant cell wall components. About 5% of the genes were differentially expressed in at least one condition. Analysis of upregulated cell wall-degrading enzymes highlights a sharp growth medium-specific adaptation process. In particular, the nature of the polysaccharides available for fungal growth induced a specific transcriptional response aiming at the targeted enzymatic degradation of the given polysaccharides.

Nanoclays for Lipase Immobilization: Biocatalyst Characterization and Activity in Polyester Synthesis

The immobilization of Candida antarctica lipase B (CALB) was performed by physical adsorption on both neat and organo-modified forms of sepiolite and montmorillonite. The influence of different parameters, e.g., solvent, enzyme loading, cross-linking, and type of clay support, on immobilization efficiency and catalyst hydrolytic activity has been investigated. The highest hydrolytic activities were obtained for CALB immobilized on organo-modified clay minerals, highlighting the beneficial effect of organo-modification. The esterification activity of these CALB/organoclay catalysts was also tested in the ring-opening polymerization of ε-caprolactone. The polymerization kinetics observed for clay-immobilized catalysts confirmed that CALB adsorbed on organo-modified montmorillonite (CALB/MMTMOD) was the highest-performing catalytic system.

The characterisation of xyloglucanase inhibitors from Humulus lupulus.

Phytopathogenic fungi secrete a powerful arsenal of enzymes that are potentially active against each polysaccharide component of the plant cell wall. To defend themselves, plants synthetise a variety of molecules that inhibit the activity of cell wall-degrading enzymes. Xyloglucan-specific endoglucanase inhibitor proteins (XEGIPs) act specifically against the members of fungal glycoside hydrolase family 12 (GH12 in the CAZy database). In the present study, we describe the identification of three XEGIP homologues from hop (Humulus lupulus L.). When incubating each of the recombinant inhibitors with an enzymatic cocktail from Aspergillus aculeatus (Viscozyme®), the xyloglucan-degrading endoglucanase activity decreased to 15% and 5% for HlXEGIP1 and HlXEGIP2, respectively, whereas no inhibition of the Viscozyme® enzymes was observed for the third (also called HlXEGIP homologue 3, or HlXEGIPh3). Fungal enzymatic cocktails from 20 different species also showed xyloglucan-degrading endoglucanase activities, and most of them were inhibited by HlXEGIP1 and -2. Furthermore, a real time RT-PCR analysis revealed variations in the spatial distribution of the genes encoding the three inhibitors and differential expression during development and (a) biotic stress. The role of XEGIPs in the plant-fungus interaction is discussed, and a model suggesting a distinct role of these XEGIP homologues is proposed: HlXEGIP1 may act in cases of abiotic stress, while HlXEGIP2 reacts to biotic stress, and physiological development may be influenced by HlXEGIPh3.

Competing Plant Cell Wall Digestion Recalcitrance by Using Fungal Substrate – Adapted Enzyme Cocktails

1.1 General needs for energy General needs for energy are still increasing. In 2000, the energy provided worldwide was 10 Gt of oil equivalent (Gtoe) and the demand is forecasted to be around 15 Gtoe for 2020 (source: Energy Information Administration [EIA], 2002, as cited in Scragg, 2005). During the 20th century, coal proportion in energy supply decreased whereas oil and gas increased drastically. First after the 1973 oil crisis and afterwards periodically depending on oil prices, developments for producing energy by new ways were considered. In the last decade, the depletion of fossil energy sources appeared as a reality although exhaustion time remains highly controversial. Currently, it is clear that considerable efforts to promote alternative sources of energy are driven by both environmental concern (limiting fuel by-products emissions) and economic necessity linked to the fossil fuel depletion.