Oxidation of model lipids representative for main paper pulp lipophilic extractives by the laccase-mediator system.

Several model lipids representative for main paper pulp lipophilic extractives - including alkanes, fatty alcohols, fatty acids, resin acids, free and esterified sterols, and triglycerides – were treated with Pycnoporus cinnabarinus laccase in the presence of 1-hydroxybenzotriazole as mediator. The reaction products were analyzed by GC and GC-MS. The laccase alone decreased the amount of some unsaturated lipids, however, the most rapid and extensive lipid modification was obtained with the laccase-mediator system. Most unsaturated lipids were largely oxidized and the dominant oxidation products detected were epoxy and hydroxy-fatty acids from fatty acids, and free and esterified 7-ketosterols and steroid ketones from sterols and sterol esters. In contrast, saturated lipids were not modified, although some of them were oxidized when the enzymatic reactions were carried out in the presence of unsaturated lipids. The results obtained are discussed in the context of enzymatic control of pitch deposits, to explain the removal of lipid mixtures during laccase-mediator treatment of different pulp types.This study was funded by the BIORENEW EU-project (NMP2-CT-2006-026456) and the Spanish MEC (BIO2007-28719-E). Beldem (Andenne, Belgium) is acknowledged for laccase supply. S.M. thanks the Spanish CSIC and CELESA for an I3P contract and J.R thanks the CSIC for an I3P fellowship.Peer reviewe

Conserved white-rot enzymatic mechanism for wood decay in the Basidiomycota genus Pycnoporus

White-rot (WR) fungi are pivotal decomposers of dead organic matter in forest ecosystems and typically use a large array of hydrolytic and oxidative enzymes to deconstruct lignocellulose. However, the extent of lignin and cellulose degradation may vary between species and wood type. Here, we combined comparative genomics, transcriptomics and secretome proteomics to identify conserved enzymatic signatures at the onset of wood-decaying activity within the Basidiomycota genus Pycnoporus. We observed a strong conservation in the genome structures and the repertoires of protein-coding genes across the four Pycnoporus species described to date, despite the species having distinct geographic distributions. We further analysed the early response of P. cinnabarinus, P. coccineus and P. sanguineus to diverse (ligno)-cellulosic substrates. We identified a conserved set of enzymes mobilized by the three species for breaking down cellulose, hemicellulose and pectin. The co-occurrence in the exo-proteomes of H2O2-producing enzymes with H2O2-consuming enzymes was a common feature of the three species, although each enzymatic partner displayed independent transcriptional regulation. Finally, cellobiose dehydrogenase-coding genes were systematically co-regulated with at least one AA9 lytic polysaccharide monooxygenase gene, indicative of enzymatic synergy in vivo. This study highlights a conserved core white-rot fungal enzymatic mechanism behind the wood-decaying process.Peer reviewe

Etude de nouvelles oxydo-réductases impliquées dans la dégradation de la biomasse végétale chez les champignons du genre Pycnoporus (de l'expression des gènes aux applications biothechnologiques)

Cette étude a pour objectif la mise en évidence, chez les basidiomycetes du genre Pycnoporus, de nouvelles oxydo-réductases impliquées dasn la dégradation de la biomasse végétale: de l'expression des gènes aux applications biotechnologiques. Les champs d'application visés concernent essentiellement le domaine de la chimie verte, dans le cadre du projet européen BIORENEW. Le travail s'est articulé autour de trois axes principaux. Le premier a concerné l'exploration de la biodiversité naturelle en particulier tropicale, pour la sélection de souches productrices de nouvelles laccases de haut potentiel d'oxydo-réduction. Le gène codant pour la laccase Lac1 chez Pycnoporus a été utilisé comme marqueur moléculaire d'identification et de relation phylogénie-fonction, mettant en évidence une distribution des souches fortement corrélée avec leur écozone. Le deuxième axe a porté sur l'isolement de trois nouvelles laccases issues de P.sanguineus et P. coccineus qui exhibent des caractéristiques biochimiques complémentaires: haute thermostabilité, résistance aux solvants, au pH, constantes catalytiques et potentiels rédox élevés. Ces enzymes constituent de bons modèles pour des applications en biotechnologies blanches:décoloration de colorants polyphénoliques, oxydation de composés modèles de type lignine non-phénolique, oligomérisation de flavonoides naturels adaptés aux applications cosmétiques et pharmaceutiques. Enfin, dans le cadre de l'annotation du génome des souches monocaryotiques P. cinnabarinus BRFM 137 et P; sanguineus BRFM 1264, dont le séquençage a été réalisé par notre Unité, un regard tout à fait nouveau est porté sur le système lignolytique du genre Pycnoporus, longtemps décrit comme produisant que de la laccase comme enzyme du système lignolytique. Pour la première fois, nous avons montré la présence de gènes codant pour tout l'arsenal enzymatique de dégradation des lignines, c'est à dire plusieurs laccases mais surtout de nombreuses peroxydases et des enzymes auxilliaires génératrices d'H2O2 comme les glyoxal oxydases. Ces nouvelles enzymes ont été caractérisées in silico. Pour la première fois également, la sécrétion effective de peroxydases, de glyoxal oxydases et d'autres FOLymes dans nos conditions de culture a également pu être démontrée par analyse protéomique.The purpose of this work was to prospect, in the genus Pycnoporus, for new oxido-reductases involved in the degradation of lignocellulosic biomass: from gene expression to biotechnological applications. This research was conducted in the framework of green chemistry applications according to BIORENEW European Project. The study was divided in three main research axes. Firstly, the exploration of natural biodiversity, especially tropical biodiversity, for the selection of new high redox potential-laccase producing strains. These strains were repositionned in a context of phylogenomic/function through the lac1 gene. Molecular clustering based on lac1 sequences enabled the distribution of P. sanguineus and P. coccineus through four distinct, well supported clades and subclades. This distribution was highly correlated with ecozones. The second part of the work deals with the biochemical and molecular characterization of three novel laccases from P. coccineus and P. sanguineus, and their applicability on natural or model phenolic substrates. The three laccases showed complementary biochemical features: high thermo- and pH stability, high catalytic efficiency and resistance to organic solvents. The three novel laccases proved to be suitable models for white biotechnology processes: polyphenolic dye decolourization, non-phenolic lignin model compound oxidation, and synthesis of new oligomers from natural flavonoids suitable for cosmetic or pharmaceutical applications. Finally, annotation of genomic data from the monocaryotic strains P. cinnabarinus BRFM 137 and P. sanguineus BRFM 1264 (genomes sequenced by the UMR1163 BCF ) was performed for lignolytic enzymes. For the first time, new oxidases (peroxidases, glyoxal oxidases and other FOLymes) were evidenced in Pycnoporus and in silico characterized. Moreover, the active secretion of several of these enzymes has been demonstrated in our culture conditions by 1D-proteomic analysisAIX-MARSEILLE1-Bib.electronique (130559902) / SudocSudocFranceF

Plant wastes and sustainable refineries: What can we learn from fungi?

International audienceThe valorization of plant wastes allows access to renewable carbon feedstocks without increasing the demand for plant biomass production. Plant wastes are the non-edible residues and waste streams from agriculture, agroindustry and forestry. The chemical diversity and recalcitrance to degradation of such wastes challenge our ability to transform and valorize these resources into value-added compounds. Fungi that thrive on plant tissues have gained a huge diversity of enzymatic toolkits for the finely-tuned degradation of glycan and lignin polymers. Our knowledge on the enzymatic systems developed by fungi now guides innovations for plant waste bioprocessing. Here, we provide an overview of the most recent findings in the hydrolytic and oxidative systems used by fungi for the degradation of recalcitrant plant polymers. We present recent promising success in applying fungal enzymes or fungal fermentations on plant wastes, and discuss the forthcoming developments that could reinforce fungal biotechnology entering a variety of industrial applications

Challenges and advances in biotechnological approaches for the synthesis of canolol and other vinylphenols from biobased p-hydroxycinnamic acids: a review

International audienceAbstract p- Hydroxycinnamic acids, such as sinapic, ferulic, p -coumaric and caffeic acids, are among the most abundant phenolic compounds found in plant biomass and agro-industrial by-products (e.g. cereal brans, sugar-beet and coffee pulps, oilseed meals). These p- hydroxycinnamic acids, and their resulting decarboxylation products named vinylphenols (canolol, 4-vinylguaiacol, 4-vinylphenol, 4-vinylcatechol), are bioactive molecules with many properties including antioxidant, anti-inflammatory and antimicrobial activities, and potential applications in food, cosmetic or pharmaceutical industries. They were also shown to be suitable precursors of new sustainable polymers and biobased substitutes for fine chemicals such as bisphenol A diglycidyl ethers. Non-oxidative microbial decarboxylation of p -hydroxycinnamic acids into vinylphenols involves cofactor-free and metal-independent phenolic acid decarboxylases (EC 4.1.1 carboxyl lyase family). Historically purified from bacteria ( Bacillus, Lactobacillus , Pseudomonas , Enterobacter genera) and some yeasts (e.g. Brettanomyces or Candida ), these enzymes were described for the decarboxylation of ferulic and p -coumaric acids into 4-vinylguaiacol and 4-vinylphenol, respectively. The catalytic mechanism comprised a first step involving p -hydroxycinnamic acid conversion into a semi-quinone that then decarboxylated spontaneously into the corresponding vinyl compound, in a second step. Bioconversion processes for synthesizing 4-vinylguaiacol and 4-vinylphenol by microbial decarboxylation of ferulic and p -coumaric acids historically attracted the most research using bacterial recombinant phenolic acid decarboxylases (especially Bacillus enzymes) and the processes developed to date included mono- or biphasic systems, and the use of free- or immobilized cells. More recently, filamentous fungi of the Neolentinus lepideus species were shown to natively produce a more versatile phenolic acid decarboxylase with high activity on sinapic acid in addition to the others p-hydroxycinnamic acids, opening the way to the production of canolol by biotechnological processes applied to rapeseed meal. Few studies have described the further microbial/enzymatic bioconversion of these vinylphenols into valuable compounds: (i) synthesis of flavours such as vanillin, 4-ethylguaiacol and 4-ethylphenol from 4-vinylguaiacol and 4-vinylphenol, (ii) laccase-mediated polymer synthesis from canolol, 4-vinylguaiacol and 4-vinylphenol

Basidiomycetes as new biotechnological tools to generate natural aromatic flavours for the food industry

International audienc

Basidiomycetes as new biotechnological tools to generate natural aromatic flavours for the food industry

International audienc

Decolourization of black oxidized olive-mill wastewater by a new tannase-producing Aspergillus flavus strain isolated from soil

International audienceBy contaminating a Tunisian soil with black oxidized and sterilized olive-mill wastewaters (OMW), 30 new indigenous fungal soil strains able to overcome the OMW toxicity could be directly selected. Ten of the fungal strains previously isolated were screened for their capability to grow in a liquid culture medium containing oxidized OMW as the only source of carbon and energy. According to these preliminary tests, strain F2 showed the best capability of removing black colour and COD (chemical oxygen demand) and was further identified as Aspergillus flavus. After optimization of batch-liquid culture conditions in the presence of oxidized OMW, the time course of biomass and enzyme production by A. flavus F2 was followed in relation to colour and COD removal. A. flavus F2 could efficiently decolourize and detoxify the black oxidized OMW (58 and 46% of colour and COD removal, respectively, after 6 days of cultivation), concomitantly with the production of tannase (8000 UI/l on day 3)

Rapeseed and sunflower meal: a review on biotechnology status and challenges

Rapeseed and sunflower are two of the world's major oilseeds. Rapeseed and sunflower meal (RSM and SFM), the by-products of oil extraction, are produced in large quantities. They are mainly composed of proteins, lignocellulosic fibres and minerals. They were initially used as a protein complement in animal feed rations and sometimes as fertilizer or as combustible source. More recently, new alternatives to these traditional uses have been developed that draw on the structure and physicochemical properties of RSM and SFM, which are plentiful sources of nitrogen and carbon nutrients. This feature, together with their cheapness and ready availability, supports the cultivation of various microorganisms in both submerged cultures and solid-state fermentation. Recent studies have thus emphasized the potential utilisation of RSM and SFM in fermentative processes, including saccharification and production of enzymes, antibiotics, antioxidants and other bio-products, opening new challenging perspectives in white biotechnology applications

Fonctionnalisation des composés végétaux feruloylés en vanilline par les basidiomycètes

chap. 13National audienc