Mireille Haon

A two-step bioconversion process for vanillin production from ferulic acid combining Aspergillus niger and Pycnoporus cinnabarinus.

A two-step bioconversion process of ferulic acid to vanillin was elaborated combining two filamentous fungi, Aspergillus niger and Pycnoporus cinnabarinus. In the first step, A. niger transformed ferulic acid to vanillic acid and in the second step vanillic acid was reduced to vanillin by P. cinnabarinus. Ferulic acid metabolism by A. niger occurred essentially via the propenoic chain degradation to lead to vanillic acid, which was subsequently decarboxylated to methoxyhydroquinone. In 3-day-old cultures of P. cinnabarinus supplied with vanillic-acid-enriched culture medium from A. niger as precursor source, vanillin was successfully produced. In order to improve the yields of the process, sequential additions of precursors were performed. Vanillic acid production by A. niger from ferulic acid reached 920 mg1-1 with a molar yield of 88% and vanillin production by P. cinnabarinus from vanillic acid attained 237 mg1-1 with a molar yield of 22%. However, the vanillic acid oxidative system producing methoxyhydroquinone was predominant in P. cinnabarinus cultures, which explained the relatively low level in vanillin.

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

BackgroundFilamentous 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.ResultsIn 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.ConclusionThis 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.

Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserina.

BackgroundThe understanding of enzymatic polysaccharide degradation has progressed intensely in the past few years with the identification of a new class of fungal-secreted enzymes, the lytic polysaccharide monooxygenases (LPMOs) that enhance cellulose conversion. In the fungal kingdom, saprotrophic fungi display a high number of genes encoding LPMOs from family AA9 but the functional relevance of this redundancy is not fully understood.ResultsIn this study, we investigated a set of AA9 LPMOs identified in the secretomes of the coprophilous ascomycete Podospora anserina, a biomass degrader of recalcitrant substrates. Their activity was assayed on cellulose in synergy with the cellobiose dehydrogenase from the same organism. We showed that the total release of oxidized oligosaccharides from cellulose was higher for PaLPMO9A, PaLPMO9E, and PaLPMO9H that harbored a carbohydrate-binding module from the family CBM1. Investigation of their regioselective mode of action revealed that PaLPMO9A and PaLPMO9H oxidatively cleaved at both C1 and C4 positions while PaLPMO9E released only C1-oxidized products. Rapid cleavage of cellulose was observed using PaLPMO9H that was the most versatile in terms of substrate specificity as it also displayed activity on cello-oligosaccharides and β-(1,4)-linked hemicellulose polysaccharides (e.g., xyloglucan, glucomannan).ConclusionsThis study provides insights into the mode of cleavage and substrate specificities of fungal AA9 LPMOs that will facilitate their application for the development of future biorefineries.

Podospora anserina Hemicellulases Potentiate the Trichoderma reesei Secretome for Saccharification of Lignocellulosic Biomass

ABSTRACT 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.

Feruloyl esterase from Aspergillus niger a comparison of the production in solid state and submerged fermentation

Solid state fermentation (SSF) culture conditions were investigated for the production of feruloyl esterase by Aspergillus niger I-1472 and compared with submerged culture conditions. Sugar beet pulp was tested for its ability to be used both as solid support and/or carbon substrate. Mycelial growth was monitored through ergosterol measurements. Under submerged culture conditions, A. niger I-1472 produced esterase active on methyl ester of cinnamic acids, principally methyl ferulate and methyl sinapinate. Under SSF culture conditions, the enzymic profile was different since significant esterase activities using methyl caffeate and methyl p-coumarate as substrate were detected, showing the presence of at least two different proteins. Northern blot analysis of the mycelium clearly indicated the expression of feruloyl esterase gene in both conditions.

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

BackgroundSaprophytic 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.ResultsThe 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.ConclusionsWith 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.

An attempt to channel the transformation of vanillic acid into vanillin by controlling methoxyhydroquinone formation in Pycnoporus cinnabarinus with cellobiose

Abstract The effects of adding cellobiose on the transformation of vanillic acid to vanillin by two strains of Pycnoporus cinnabarinus MUCL39532 and MUCL38467 were studied. When maltose was used as the carbon source in the culture medium, very high levels of methoxyhydroquinone were formed from vanillic acid. When cellobiose was used as the carbon source and/or added to the culture medium of P. cinnabarinus strains on day 3 just before vanillic acid was added, it channelled the vanillic acid metabolism via the reductive route leading to vanillin. Adding 3.5 g l−1 cellobiose to 3-day-old maltose cultures of P. cinnabarinus MUCL39532 and 2.5 g l−1 cellobiose to 3-day-old cellobiose cultures of P. cinnabarinus MUCL38467, yielded 510 mg l−1 and 560 mg l−1 vanillin with a molar yield of 50.2 % and 51.7 % respectively. Cellobiose may either have acted as an easily metabolizable carbon source, required for the reductive pathway to occur, or as an inducer of cellobiose:quinone oxidoreductase, which is known to inhibit vanillic acid decarboxylation.

Exploring the Natural Fungal Biodiversity of Tropical and Temperate Forests toward Improvement of Biomass Conversion

ABSTRACT 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.

By-passing of unwanted vanillyl alcohol formation using selective adsorbents to improve vanillin production with Phanerochaete chrysosporium

Phanerochaete chrysosporium has been found to preferentially metabolize vanillic acid using a reduction pathway which leads to vanillyl alcohol via vanillin. In order to prevent vanillin transformation into unwanted vanillyl alcohol, the ability of different adsorbents to adsorb vanillin was investigated. XAD-2 resin was shown to be very efficient and selective for vanillin. When applied to the culture medium of P. chrysosporium, the resin trapped the vanillin, which prevented the transformation of vanillin into vanillyl alcohol.

First structural insights into α-L-arabinofuranosidases from the two GH62 glycoside hydrolase subfamilies

Background: α-l-Arabinofuranosidases hydrolyze arabinofuranosyl side chains from xylans. Results: The first crystal structures of two fungal α-l-arabinofuranosidases representing two distinct subfamilies from the glycoside hydrolase GH62 family are presented. The examination of these unveils specificity determinants. Conclusion: The structures of complexes with arabinose and cellotriose provide preliminary insight into substrate recognition and catalysis. Significance: This work provides the first structural description members of the GH62 family. α-l-Arabinofuranosidases are glycoside hydrolases that specifically hydrolyze non-reducing residues from arabinose-containing polysaccharides. In the case of arabinoxylans, which are the main components of hemicellulose, they are part of microbial xylanolytic systems and are necessary for complete breakdown of arabinoxylans. Glycoside hydrolase family 62 (GH62) is currently a small family of α-l-arabinofuranosidases that contains only bacterial and fungal members. Little is known about the GH62 mechanism of action, because only a few members have been biochemically characterized and no three-dimensional structure is available. Here, we present the first crystal structures of two fungal GH62 α-l-arabinofuranosidases from the basidiomycete Ustilago maydis (UmAbf62A) and ascomycete Podospora anserina (PaAbf62A). Both enzymes are able to efficiently remove the α-l-arabinosyl substituents from arabinoxylan. The overall three-dimensional structure of UmAbf62A and PaAbf62A reveals a five-bladed β-propeller fold that confirms their predicted classification into clan GH-F together with GH43 α-l-arabinofuranosidases. Crystallographic structures of the complexes with arabinose and cellotriose reveal the important role of subsites +1 and +2 for sugar binding. Intriguingly, we observed that PaAbf62A was inhibited by cello-oligosaccharides and displayed binding affinity to cellulose although no activity was observed on a range of cellulosic substrates. Bioinformatic analyses showed that UmAbf62A and PaAbf62A belong to two distinct subfamilies within the GH62 family. The results presented here provide a framework to better investigate the structure-function relationships within the GH62 family.