Large-scale phenotyping of 1,000 fungal strains for the degradation of non-natural, industrial compounds.

Fungal biotechnology is set to play a keystone role in the emerging bioeconomy, notably to address pollution issues arising from human activities. Because they preserve biological diversity, Biological Resource Centres are considered as critical infrastructures to support the development of biotechnological solutions. Here, we report the first large-scale phenotyping of more than 1,000 fungal strains with evaluation of their growth and degradation potential towards five industrial, human-designed and recalcitrant compounds, including two synthetic dyes, two lignocellulose-derived compounds and a synthetic plastic polymer. We draw a functional map over the phylogenetic diversity of Basidiomycota and Ascomycota, to guide the selection of fungal taxa to be tested for dedicated biotechnological applications. We evidence a functional diversity at all taxonomic ranks, including between strains of a same species. Beyond demonstrating the tremendous potential of filamentous fungi, our results pave the avenue for further functional exploration to solve the ever-growing issue of ecosystems pollution.

Gene family expansions and transcriptome signatures uncover fungal adaptations to wood decay.

Because they comprise some of the most efficient wood-decayers, Polyporales fungi impact carbon cycling in forest environment. Despite continuous discoveries on the enzymatic machinery involved in wood decomposition, the vision on their evolutionary adaptation to wood decay and genome diversity remains incomplete. We combined the genome sequence information from 50 Polyporales species, including 26 newly sequenced genomes and sought for genomic and functional adaptations to wood decay through the analysis of genome composition and transcriptome responses to different carbon sources. The genomes of Polyporales from different phylogenetic clades showed poor conservation in macrosynteny, indicative of genome rearrangements. We observed different gene family expansion/contraction histories for plant cell wall degrading enzymes in core polyporoids and phlebioids and captured expansions for genes involved in signalling and regulation in the lineages of white rotters. Furthermore, we identified conserved cupredoxins, thaumatin-like proteins and lytic polysaccharide monooxygenases with a yet uncharacterized appended module as new candidate players in wood decomposition. Given the current need for enzymatic toolkits dedicated to the transformation of renewable carbon sources, the observed genomic diversity among Polyporales strengthens the relevance of mining Polyporales biodiversity to understand the molecular mechanisms of wood decay.

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.

Screening of five marine-derived fungal strains for their potential to produce oxidases with laccase activities suitable for biotechnological applications.

BACKGROUND: Environmental pollution is one of the major problems that the world is facing today. Several approaches have been taken, from physical and chemical methods to biotechnological strategies (e.g. the use of oxidoreductases). Oxidative enzymes from microorganisms offer eco-friendly, cost-effective processes amenable to biotechnological applications, such as in industrial dye decolorization. The aim of this study was to screen marine-derived fungal strains isolated from three coastal areas in Tunisia to identify laccase-like activities, and to produce and characterize active cell-free supernatants of interest for dye decolorization. RESULTS: Following the screening of 20 fungal strains isolated from the harbors of Sfax and Monastir (Tunisia), five strains were identified that displayed laccase-like activities. Molecular-based taxonomic approaches identified these strains as belonging to the species Trichoderma asperellum, Stemphylium lucomagnoense and Aspergillus nidulans. Among these five isolates, one T. asperellum strain (T. asperellum 1) gave the highest level of secreted oxidative activities, and so was chosen for further studies. Optimization of the growth medium for liquid cultures was first undertaken to improve the level of laccase-like activity in culture supernatants. Finally, the culture supernatant of T. asperellum 1 decolorized different synthetic dyes belonging to diverse dye families, in the presence or absence of 1-hydroxybenzotriazole (HBT) as a mediator. CONCLUSIONS: The optimal growth conditions to produce laccase-like active cell-free supernatants from T. asperellum 1 were 1.8 mM CuSO4 as an inducer, 1% NaCl to mimic a seawater environment and 3% sucrose as a carbon source. The culture supernatant of T. asperellum 1 effectively decolorized different synthetic dyes belonging to diverse chemical classes, and the presence of HBT as a mediator improved the decolorization process.

Lavender- and lavandin-distilled straws: an untapped feedstock with great potential for the production of high-added value compounds and fungal enzymes.

BACKGROUND: Lavender (Lavandula angustifolia) and lavandin (a sterile hybrid of L. angustifolia × L. latifolia) essential oils are among those most commonly used in the world for various industrial purposes, including perfumes, pharmaceuticals and cosmetics. The solid residues from aromatic plant distillation such as lavender- and lavandin-distilled straws are generally considered as wastes, and consequently either left in the fields or burnt. However, lavender- and lavandin-distilled straws are a potentially renewable plant biomass as they are cheap, non-food materials that can be used as raw feedstocks for green chemistry industry. The objective of this work was to assess different pathways of valorization of these straws as bio-based platform chemicals and fungal enzymes of interest in biorefinery. RESULTS: Sugar and lignin composition analyses and saccharification potential of the straw fractions revealed that these industrial by-products could be suitable for second-generation bioethanol prospective. The solvent extraction processes, developed specifically for these straws, released terpene derivatives (e.g. τ-cadinol, ß-caryophyllene), lactones (e.g. coumarin, herniarin) and phenolic compounds of industrial interest, including rosmarinic acid which contributed to the high antioxidant activity of the straw extracts. Lavender and lavandin straws were also suitable inducers for the secretion of a wide panel of lignocellulose-acting enzymes (cellulases, hemicellulases and oxido-reductases) from the white-rot model fungus Pycnoporus cinnabarinus. Interestingly, high amounts of laccase and several lytic polysaccharide monooxygenases were identified in the lavender and lavandin straw secretomes using proteomics. CONCLUSIONS: The present study demonstrated that the distilled straws of lavender and lavandin are lignocellulosic-rich materials that can be used as raw feedstocks for producing high-added value compounds (antioxidants, aroma) and fungal oxidative enzymes, which represent opportunities to improve the decomposition of recalcitrant lignocellulose into biofuel. Hence, the structure and the physico-chemical properties of these straws clearly open new perspectives for use in biotechnological processes involving especially filamentous fungi. These approaches represent sustainable strategies to foster the development of a local circular bioeconomy.

Integrative visual omics of the white-rot fungus Polyporus brumalis exposes the biotechnological potential of its oxidative enzymes for delignifying raw plant biomass.

BACKGROUND: Plant biomass conversion for green chemistry and bio-energy is a current challenge for a modern sustainable bioeconomy. The complex polyaromatic lignin polymers in raw biomass feedstocks (i.e., agriculture and forestry by-products) are major obstacles for biomass conversions. White-rot fungi are wood decayers able to degrade all polymers from lignocellulosic biomass including cellulose, hemicelluloses, and lignin. The white-rot fungus Polyporus brumalis efficiently breaks down lignin and is regarded as having a high potential for the initial treatment of plant biomass in its conversion to bio-energy. Here, we describe the extraordinary ability of P. brumalis for lignin degradation using its enzymatic arsenal to break down wheat straw, a lignocellulosic substrate that is considered as a biomass feedstock worldwide. RESULTS: We performed integrative multi-omics analyses by combining data from the fungal genome, transcriptomes, and secretomes. We found that the fungus possessed an unexpectedly large set of genes coding for Class II peroxidases involved in lignin degradation (19 genes) and GMC oxidoreductases/dehydrogenases involved in generating the hydrogen peroxide required for lignin peroxidase activity and promoting redox cycling of the fungal enzymes involved in oxidative cleavage of lignocellulose polymers (36 genes). The examination of interrelated multi-omics patterns revealed that eleven Class II Peroxidases were secreted by the fungus during fermentation and eight of them where tightly co-regulated with redox cycling enzymatic partners. CONCLUSION: As a peculiar feature of P. brumalis, we observed gene family extension, up-regulation and secretion of an abundant set of versatile peroxidases and manganese peroxidases, compared with other Polyporales species. The orchestrated secretion of an abundant set of these delignifying enzymes and redox cycling enzymatic partners could contribute to the delignification capabilities of the fungus. Our findings highlight the diversity of wood decay mechanisms present in Polyporales and the potentiality of further exploring this taxonomic order for enzymatic functions of biotechnological interest.

Essential oils and distilled straws of lavender and lavandin: a review of current use and potential application in white biotechnology.

The Lavandula genus, which includes lavender (Lavandula angustifolia) and lavandin (L. angustifolia × Lavandula latifolia), is cultivated worldwide for its essential oils, which find applications in perfumes, cosmetics, food processing and, more recently, in aromatherapy products. The chemical composition of lavender and lavandin essential oils, usually produced by steam distillation from the flowering stems, is characterized by the presence of terpenes (e.g. linalool and linalyl acetate) and terpenoids (e.g. 1,8-cineole), which are mainly responsible for their characteristic flavour and their biological and therapeutic properties. Lavender and lavandin distilled straws, the by-products of oil extraction, were traditionally used for soil replenishment or converted to a fuel source. They are mineral- and carbon-rich plant residues and, therefore, a cheap, readily available source of valuable substances of industrial interest, especially aroma and antioxidants (e.g. terpenoids, lactones and phenolic compounds including coumarin, herniarin, α-bisabolol, rosmarinic and chlorogenic acids). Accordingly, recent studies have emphasized the possible uses of lavender and lavandin straws in fermentative or enzymatic processes involving various microorganisms, especially filamentous fungi, for the production of antimicrobials, antioxidants and other bioproducts with pharmaceutical and cosmetic activities, opening up new challenging perspectives in white biotechnology applications.

Fast solubilization of recalcitrant cellulosic biomass by the basidiomycete fungus Laetisaria arvalis involves successive secretion of oxidative and hydrolytic enzymes.

BACKGROUND: Enzymatic breakdown of lignocellulosic biomass is a known bottleneck for the production of high-value molecules and biofuels from renewable sources. Filamentous fungi are the predominant natural source of enzymes acting on lignocellulose. We describe the extraordinary cellulose-deconstructing capacity of the basidiomycete Laetisaria arvalis, a soil-inhabiting fungus. RESULTS: The L. arvalis strain displayed the capacity to grow on wheat straw as the sole carbon source and to fully digest cellulose filter paper. The cellulolytic activity exhibited in the secretomes of L. arvalis was up to 7.5 times higher than that of a reference Trichoderma reesei industrial strain, resulting in a significant improvement of the glucose release from steam-exploded wheat straw. Global transcriptome and secretome analyses revealed that L. arvalis produces a unique repertoire of carbohydrate-active enzymes in the fungal taxa, including a complete set of enzymes acting on cellulose. Temporal analyses of secretomes indicated that the unusual degradation efficiency of L. arvalis relies on its early response to the carbon source, and on the finely tuned sequential secretion of several lytic polysaccharide monooxygenases and hydrolytic enzymes targeting cellulose. CONCLUSIONS: The present study illustrates the adaptation of a litter-rot fungus to the rapid breakdown of recalcitrant plant biomass. The cellulolytic capabilities of this basidiomycete fungus result from the rapid, selective and successive secretion of oxidative and hydrolytic enzymes. These enzymes expressed at critical times during biomass degradation may inspire the design of improved enzyme cocktails for the conversion of plant cell wall resources into fermentable sugars.

The genome of the white-rot fungus Pycnoporus cinnabarinus: a basidiomycete model with a versatile arsenal for lignocellulosic biomass breakdown.

BACKGROUND: Saprophytic filamentous fungi are ubiquitous micro-organisms that play an essential role in photosynthetic carbon recycling. The wood-decayer Pycnoporus cinnabarinus is a model fungus for the study of plant cell wall decomposition and is used for a number of applications in green and white biotechnology. RESULTS: The 33.6 megabase genome of P. cinnabarinus was sequenced and assembled, and the 10,442 predicted genes were functionally annotated using a phylogenomic procedure. In-depth analyses were carried out for the numerous enzyme families involved in lignocellulosic biomass breakdown, for protein secretion and glycosylation pathways, and for mating type. The P. cinnabarinus genome sequence revealed a consistent repertoire of genes shared with wood-decaying basidiomycetes. P. cinnabarinus is thus fully equipped with the classical families involved in cellulose and hemicellulose degradation, whereas its pectinolytic repertoire appears relatively limited. In addition, P. cinnabarinus possesses a complete versatile enzymatic arsenal for lignin breakdown. We identified several genes encoding members of the three ligninolytic peroxidase types, namely lignin peroxidase, manganese peroxidase and versatile peroxidase. Comparative genome analyses were performed in fungi displaying different nutritional strategies (white-rot and brown-rot modes of decay). P. cinnabarinus presents a typical distribution of all the specific families found in the white-rot life style. Growth profiling of P. cinnabarinus was performed on 35 carbon sources including simple and complex substrates to study substrate utilization and preferences. P. cinnabarinus grew faster on crude plant substrates than on pure, mono- or polysaccharide substrates. Finally, proteomic analyses were conducted from liquid and solid-state fermentation to analyze the composition of the secretomes corresponding to growth on different substrates. The distribution of lignocellulolytic enzymes in the secretomes was strongly dependent on growth conditions, especially for lytic polysaccharide mono-oxygenases. CONCLUSIONS: With its available genome sequence, P. cinnabarinus is now an outstanding model system for the study of the enzyme machinery involved in the degradation or transformation of lignocellulosic biomass.

FunGene-DB: a web-based tool for Polyporales strains authentication.

Polyporales are extensively studied wood-decaying fungi with applications in white and green biotechnologies and in medicinal chemistry. We developed an open-access, user-friendly, bioinformatics tool named FunGene-DB (http://www.fungene-db.org). The goal was to facilitate the molecular authentication of Polyporales strains and fruit-bodies, otherwise subjected to morphological studies. This tool includes a curated database that contains ITS1-5.8S-ITS2 rDNA genes screened through a semi-automated pipeline from the International Nucleotide Sequence Database (INSD), and the similarity search BLASTn program. Today, the web-accessible database compiles 2379 accepted sequences, among which 386 were selected as reference sequences (most often fully identified ITS sequences for which a voucher, strain or specimen, has been deposited in a public-access collection). The restriction of the database to one reference sequence per species (or per clade for species complex) allowed most often unequivocal analysis. We conclude that FunGene-DB is a promising tool for molecular authentication of Polyporales. It should be especially useful for scientists who are not expert mycologists but who need to check the identity of strains (e.g. for culture collections, for applied microbiology).

Exploring the natural fungal biodiversity of tropical and temperate forests toward improvement of biomass conversion.

In this study, natural fungal diversity in wood-decaying species was explored for biomass deconstruction. In 2007 and 2008, fungal isolates were collected in temperate forests mainly from metropolitan France and in tropical forests mainly from French Guiana. We recovered and identified 74 monomorph cultures using morphological and molecular identification tools. Following production of fungal secretomes under inductive conditions, we evaluated the capacity of these fungal strains to potentiate a commercial Trichoderma reesei cellulase cocktail for the release of soluble sugars from biomass. The secretome of 19 isolates led to an improvement in biomass conversion of at least 23%. Of the isolates, the Trametes gibbosa BRFM 952 (Banque de Ressources Fongiques de Marseille) secretome performed best, with 60% improved conversion, a feature that was not universal to the Trametes and related genera. Enzymatic characterization of the T. gibbosa BRFM 952 secretome revealed an unexpected high activity on crystalline cellulose, higher than that of the T. reesei cellulase cocktail. This report highlights the interest in a systematic high-throughput assessment of collected fungal biodiversity to improve the enzymatic conversion of lignocellulosic biomass. It enabled the unbiased identification of new fungal strains issued from biodiversity with high biotechnological potential.

Post-genomic analyses of fungal lignocellulosic biomass degradation reveal the unexpected potential of the plant pathogen Ustilago maydis.

BACKGROUND: Filamentous fungi are potent biomass degraders due to their ability to thrive in ligno(hemi)cellulose-rich environments. During the last decade, fungal genome sequencing initiatives have yielded abundant information on the genes that are putatively involved in lignocellulose degradation. At present, additional experimental studies are essential to provide insights into the fungal secreted enzymatic pools involved in lignocellulose degradation. RESULTS: In this study, we performed a wide analysis of 20 filamentous fungi for which genomic data are available to investigate their biomass-hydrolysis potential. A comparison of fungal genomes and secretomes using enzyme activity profiling revealed discrepancies in carbohydrate active enzymes (CAZymes) sets dedicated to plant cell wall. Investigation of the contribution made by each secretome to the saccharification of wheat straw demonstrated that most of them individually supplemented the industrial Trichoderma reesei CL847 enzymatic cocktail. Unexpectedly, the most striking effect was obtained with the phytopathogen Ustilago maydis that improved the release of total sugars by 57% and of glucose by 22%. Proteomic analyses of the best-performing secretomes indicated a specific enzymatic mechanism of U. maydis that is likely to involve oxido-reductases and hemicellulases. CONCLUSION: This study provides insight into the lignocellulose-degradation mechanisms by filamentous fungi and allows for the identification of a number of enzymes that are potentially useful to further improve the industrial lignocellulose bioconversion process.

A thermostable GH45 endoglucanase from yeast: impact of its atypical multimodularity on activity.

BACKGROUND: The gene encoding an atypical multi-modular glycoside hydrolase family 45 endoglucanase bearing five different family 1 carbohydrate binding modules (CBM1), designated PpCel45A, was identified in the Pichia pastoris GS115 genome. RESULTS: PpCel45A (full-length open reading frame), and three derived constructs comprising (i) the catalytic module with its proximal CBM1, (ii) the catalytic module only, and (iii) the five CBM1 modules without catalytic module, were successfully expressed to high yields (up to 2 grams per litre of culture) in P. pastoris X33. Although the constructs containing the catalytic module displayed similar activities towards a range of glucans, comparison of their biochemical characteristics revealed striking differences. We observed a high thermostability of PpCel45A (Half life time of 6 h at 80°C), which decreased with the removal of CBMs and glycosylated linkers. However, both binding to crystalline cellulose and hydrolysis of crystalline cellulose and cellohexaose were substantially boosted by the presence of one CBM rather than five. CONCLUSIONS: The present study has revealed the specific features of the first characterized endo ß-1,4 glucanase from yeast, whose thermostability is promising for biotechnological applications related to the saccharification of lignocellulosic biomass such as consolidated bioprocessing.

Peculiarities of Pycnoporus species for applications in biotechnology.

The genus Pycnoporus forms a cosmopolitan group of four species belonging to the polyporoid white-rot fungi, the most representative group of homobasidiomycetes causing wood decay. Pycnoporus fungi are listed as food- and cosmetic-grade microorganisms and emerged in the early 1990s as a genus whose biochemistry, biodegradation and biotechnological properties have since been progressively detailed. First highlighted for their original metabolic pathways involved in the functionalization of plant cell wall aromatic compounds to yield high-value molecules, e.g. aromas and antioxidants, the Pycnoporus species were later explored for their potential to produce various enzymes of industrial interest, such as hydrolases and oxidases. However, the most noteworthy feature of the genus Pycnoporus is its ability to overproduce high redox potential laccase-a multi-copper extracellular phenoloxidase-as the predominant ligninolytic enzyme. A major potential use of the Pycnoporus fungi is thus to harness their laccases for various applications such as the bioconversion of agricultural by-products and raw plant materials into valuable products, the biopulping and biobleaching of paper pulp and the biodegradation of organopollutants, xenobiotics and industrial contaminants. All the studies performed in the last decade show the genus Pycnoporus to be a strong contender for white biotechnology. In this review, we describe the properties of Pycnoporus fungi in relation to their biotechnological applications and potential.

Phylogeographic relationships in the polypore fungus Pycnoporus inferred from molecular data.

The genus Pycnoporus forms a group of four species known especially for producing high redox potential laccases suitable for white biotechnology. A sample of 36 Pycnoporus strains originating from different geographical areas was studied to seek informative molecular markers for the typing of new strains in laboratory culture conditions and to analyse the phylogeographic relationships in this cosmopolitan group. ITS1-5.8S-ITS2 ribosomal DNA and partial regions of ß-tubulin and laccase lac3-1 gene were sequenced. Phylogenetic trees inferred from these sequences clearly differentiated the group of Pycnoporus cinnabarinus strains from the group of Pycnoporus puniceus strains into strongly supported clades (100% bootstrap value). Molecular clustering based on lac 3-1 sequences enabled the distribution of Pycnoporus sanguineus and Pycnoporus coccineus through four distinct, well supported clades and sub-clades. A neotropical sub-clade, grouping the P. sanguineus strains from French Guiana and Venezuela, corresponded to P. sanguineus sensu stricto. A paleotropical sub-clade, clustering the strains from Madagascar, Vietnam and New Caledonia, was defined as Pycnoporus cf. sanguineus. The Australian clade corresponded to P. coccineus sensu stricto. The Eastern Asian region clade, clustering the strains from China and Japan, formed a P. coccineus-like group. Laccase gene (lac 3-1) analysis within the Pycnoporus species can highlight enzyme functional diversity associated with biogeographical origin.

Pycnoporus laccase-mediated bioconversion of rutin to oligomers suitable for biotechnology applications.

The Pycnoporus fungi are white-rot basidiomycetes listed as food- and cosmetic-grade microorganisms. Three high redox potential laccases from Pycnoporus coccineus and Pycnoporus sanguineus were tested and compared, with the commercial Suberase® as reference, for their ability to synthesise natural active oligomers from rutin (quercetin-3-rutinoside, one of the best-known naturally occurring flavonoid glycosides). The aim of this work was to develop a process with technical parameters (solvent, temperature, reaction time and raw materials) that were easy to scale up for industrial production and compatible with cosmetic and pharmaceutical formulation guidelines. The aqueous mixture of glycerol/ethanol/buffer described in this study met this requirement and allowed the solubilisation of rutin and its oxidative bioconversion into oligomers. The four flavonoid oligomer mixtures synthesised using laccases as catalysts were analysed by high performance liquid chromatography-diode array detection-negative electrospray ionisation-multistage mass spectrometry. Their chromatographic elution profiles were compared and 16 compounds were characterised and identified as dimers and trimers of rutin. The oligorutins were different in Suberase® and Pycnoporus laccase reaction mixtures. They were evaluated for their antioxidant, anti-inflammatory and anti-ageing activities on specific enzymatic targets such as cyclooxygenase (COX-2) and human matrix metalloproteinase 3 (MMP-3). Expressed in terms of IC(50), the flavonoid oligomers displayed a 2.5- to 3-fold higher superoxide scavenging activity than monomeric rutin. Pycnoporus laccase and Suberase® oligorutins led to an inhibition of COX-2 of about 35% and 70%, respectively, while monomeric rutin showed a near-negligible inhibition effect, less than about 10%. The best results on MMP-3 activity were obtained with rutin oligomers from P. sanguineus IMB W006-2 laccase and Suberase® with about 70-75% inhibition.

Podospora anserina hemicellulases potentiate the Trichoderma reesei secretome for saccharification of lignocellulosic biomass.

To improve the enzymatic hydrolysis (saccharification) of lignocellulosic biomass by Trichoderma reesei, a set of genes encoding putative polysaccharide-degrading enzymes were selected from the coprophilic fungus Podospora anserina using comparative genomics. Five hemicellulase-encoding genes were successfully cloned and expressed as secreted functional proteins in the yeast Pichia pastoris. These novel fungal CAZymes belonging to different glycoside hydrolase families (PaMan5A and PaMan26A mannanases, PaXyn11A xylanase, and PaAbf51A and PaAbf62A arabinofuranosidases) were able to break down their predicted cognate substrates. Although PaMan5A and PaMan26A displayed similar specificities toward a range of mannan substrates, they differed in their end products, suggesting differences in substrate binding. The N-terminal CBM35 module of PaMan26A displayed dual binding specificity toward xylan and mannan. PaXyn11A harboring a C-terminal CBM1 module efficiently degraded wheat arabinoxylan, releasing mainly xylobiose as end product. PaAbf51A and PaAbf62A arabinose-debranching enzymes exhibited differences in activity toward arabinose-containing substrates. Further investigation of the contribution made by each P. anserina auxiliary enzyme to the saccharification of wheat straw and spruce demonstrated that the endo-acting hemicellulases (PaXyn11A, PaMan5A, and PaMan26A) individually supplemented the secretome of the industrial T. reesei CL847 strain. The most striking effect was obtained with PaMan5A that improved the release of total sugars by 28% and of glucose by 18%, using spruce as lignocellulosic substrate.

New chromogenic substrates for feruloyl esterases.

Indolyl and nitrophenyl 5-O-hydroxycinnamoyl-alpha-L-arabinofuranosides were prepared by chemo-enzymatic syntheses. These probes were designed as substrates to be used in assays of feruloyl esterase activity (EC 3.1.1.77). Color development in the assays only occurs when feruloyl esterase activity releases an intermediate chromogenic arabinoside that is a suitable substrate for alpha-L-arabinofuranosidase (EC 3.2.1.55), which in turn releases the free chromogenic group. The usefulness of these compounds was evaluated in both qualitative solid media-based assays and quantitative liquid assays that can be performed in microtiter plates using feruloyl esterases and arabinofuranosidases from various origins.

Gene overexpression and biochemical characterization of the biotechnologically relevant chlorogenic acid hydrolase from Aspergillus niger.

The full-length gene that encodes the chlorogenic acid hydrolase from Aspergillus niger CIRM BRFM 131 was cloned by PCR based on the genome of the strain A. niger CBS 513.88. The complete gene consists of 1,715 bp and codes for a deduced protein of 512 amino acids with a molecular mass of 55,264 Da and an acidic pI of 4.6. The gene was successfully cloned and overexpressed in A. niger to yield 1.25 g liter(-1), i.e., 330-fold higher than the production of wild-type strain A. niger CIRM BRFM131. The histidine-tagged recombinant ChlE protein was purified to homogeneity via a single chromatography step, and its main biochemical properties were characterized. The molecular size of the protein checked by mass spectroscopy was 74,553 Da, suggesting the presence of glycosylation. ChlE is assembled in a tetrameric form with several acidic isoforms with pIs of around 4.55 and 5.2. Other characteristics, such as optimal pH and temperature, were found to be similar to those determined for the previously characterized chlorogenic acid hydrolase of A. niger CIRM BRFM 131. However, there was a significant temperature stability difference in favor of the recombinant protein. ChlE exhibits a catalytic efficiency of 12.5 x 10(6) M(-1) s(-1) toward chlorogenic acid (CGA), and its ability to release caffeic acid from CGA present in agricultural by-products such as apple marc and coffee pulp was clearly demonstrated, confirming the high potential of this enzyme.

Chromogenic substrates for feruloyl esterases.

Chromogenic mono- and diferuloyl-butanetriol analogs were prepared by chemical syntheses and their efficiency was evaluated as substrates for feruloyl esterases from Aspergillus niger.