Marcel Asther

Comparative secretome analyses of two Trichoderma reesei RUT-C30 and CL847 hypersecretory strains

BackgroundDue to its capacity to produce large amounts of cellulases, Trichoderma reesei is increasingly been researched in various fields of white biotechnology, especially in biofuel production from lignocellulosic biomass. The commercial enzyme mixtures produced at industrial scales are not well characterized, and their proteinaceous components are poorly identified and quantified. The development of proteomic methods has made it possible to comprehensively overview the enzymes involved in lignocellulosic biomass degradation which are secreted under various environmental conditions.ResultsThe protein composition of the secretome produced by industrial T. reesei (strain CL847) grown on a medium promoting the production of both cellulases and hemicellulases was explored using two-dimensional electrophoresis and MALDI-TOF or LC-MS/MS protein identification. A total of 22 protein species were identified. As expected, most of them are potentially involved in biomass degradation. The 2D map obtained was then used to compare the secretomes produced by CL847 and another efficient cellulolytic T. reesei strain, Rut-C30, the reference cellulase-overproducing strain using lactose as carbon source and inducer of cellulases.ConclusionThis study provides the most complete mapping of the proteins secreted by T. reesei to date. We report on the first use of proteomics to compare secretome composition between two cellulase-overproducing strains Rut-C30 and CL847 grown under similar conditions. Comparison of protein patterns in both strains highlighted many unexpected differences between cellulase cocktails. The results demonstrate that 2D electrophoresis is a promising tool for studying cellulase production profiles, whether for industrial characterization of an entire secretome or for a more fundamental study on cellulase expression at genome-wide scale.

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.

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

Consumer preference for natural food additives has led to an increasing demand for natural aromatic compounds. An alternative production process to plant and chemical sources is the use of biotechnological methods involving microorganisms, which ensure a stable supply, quality and price. Among filamentous fungi, white-rot basidiomycetes represent an important group that generate a wide range of flavouring compounds, particularly aromatic molecules. Their biotechnological potential to produce natural aromatic flavours de novo or by biotransformation thus presents a very interesting challenge.

Effect of Tween 80 and oleic acid on ligninase production by Phanerochaete chrysosporium INA-12

Ligninase production by Phanerochaete chrysosporium INA-12 was increased when culture medium was supplemented with sorbitan polyoxyethylene monooleate (Tween 80), oleic acid alone or emulsified with Tween 80. Results showed that fatty acids contained in Tween 80 were dissimilated by P. chrysosporium INA-12 in glycerol-containing cultures. Lipase activity (EC 3.1.1.3) was detected in the culture filtrate after one day of incubation. Ligninase production was markedly enhanced and fermentation time for maximum activity was reduced in presence of exogenous oleic acid emulsified with Tween 80 (0.04%, w/v). Ligninase activity in stationary and agitated conditions were 22.4 and 19.3 nkat ml−1respectively. Agitated cultures containing Tween 80 or emulsified oleic acid converted [ring-U-14C]lignin to14CO2at a rate in proportion to ligninase activity. The effect of additives as well as the development of suitable culture conditions for ligninase production by P. chrysosporium INA-12 using submerged agitated cultures is discussed.

Highly Efficient Production of Laccase by the Basidiomycete Pycnoporus cinnabarinus

ABSTRACT An efficient transformation and expression system was developed for the industrially relevant basidiomycete Pycnoporus cinnabarinus. This was used to transform a laccase-deficient monokaryotic strain with the homologous lac1 laccase gene placed under the regulation of its own promoter or that of the SC3 hydrophobin gene or the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of Schizophyllum commune. SC3-driven expression resulted in a maximal laccase activity of 107 nkat ml−1 in liquid shaken cultures. This value was about 1.4 and 1.6 times higher in the cases of the GPD and lac1 promoters, respectively. lac1-driven expression strongly increased when 25 g of ethanol liter−1 was added to the medium. Accordingly, laccase activity increased to 1,223 nkat ml−1. These findings agree with the fact that ethanol induces laccase gene expression in some fungi. Remarkably, lac1 mRNA accumulation and laccase activity also strongly increased in the presence of 25 g of ethanol liter−1 when lac1 was expressed behind the SC3 or GPD promoter. In the latter case, a maximal laccase activity of 1,393 nkat ml−1 (i.e., 360 mg liter−1) was obtained. Laccase production was further increased in transformants expressing lac1 behind its own promoter or that of GPD by growth in the presence of 40 g of ethanol liter−1. In this case, maximal activities were 3,900 and 4,660 nkat ml−1, respectively, corresponding to 1 and 1.2 g of laccase per liter and thus representing the highest laccase activities reported for recombinant fungal strains. These results suggest that P. cinnabarinus may be a host of choice for the production of other proteins as well.

FOLy: An integrated database for the classification and functional annotation of fungal oxidoreductases potentially involved in the degradation of lignin and related aromatic compounds

The breakdown of lignin by fungi is a key step during carbon recycling in terrestrial ecosystems. This process is of great interest for green and white biotechnological applications. Given the importance of these enzymatic processes, we have classified the enzymes potentially involved in lignin catabolism into sequence-based families and integrated them in a newly developed database, designated Fungal Oxidative Lignin enzymes (FOLy). Families were defined after sequence similarity searches starting from protein sequences and validated by the convergence of results with biochemical experiments reported in the literature. The resulting database was applied as a tool for the functional annotation of genomes from different fungi, namely (i) the Basidiomycota Coprinopsis cinerea, Phanerochaete chrysosporium and Ustilago maydis and (ii) the Ascomycota Aspergillus nidulans and Trichoderma reesei. Genomic comparison of the oxidoreductases of these fungi revealed significant differences in the putative enzyme arsenals. Two Ascomycota fungal genomes were annotated and new candidate genes were identified that could be useful for lignin degradation and (or) melanin synthesis, and their function investigated experimentally. This database efforts aims at providing the means to get new insights for the understanding and biotechnological exploitation of the lignin degradation. A WWW server giving access to the routinely updated FOLy classifications of enzymes potentially involved in lignin degradation can be found at http://foly.esil.univ-mrs.fr.

Secretome analysis of Phanerochaete chrysosporium strain CIRM-BRFM41 grown on softwood

Proteomic analysis was performed to determine and differentiate the composition of the secretomes of Phanerochaete chrysosporium CIRM-BRFM41, a peroxidase hypersecretory strain grown under ligninolytic conditions and on softwood chips under biopulping conditions. Extracellular proteins from both cultures were analyzed by bidimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. A total of 37 spots were identified. The secretome in liquid synthetic medium comprised mainly peroxidases, while several wood-degrading enzymes and enzymes involved in fungal metabolism were detected in biopulping cultures on softwood. This prompted an analysis of the impact of secretome modulation in the presence of softwood chips. Biotreated wood was submitted to kraft cooking and chemical bleaching using chlorine dioxide. The fungal pre-treatment led to a significant increase in pulp yield and a better bleachability of the pulp. This bleachability improvement could be explained by the production of specific lignocellulose-degrading enzymes.

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.

A biotechnological process involving filamentous fungi to produce natural crystalline vanillin from maize bran

A new process involving the filamentous fungi Aspergillus niger and Pycnoporus cinnabarinus has been designed for the release of ferulic acid by enzymic degradation of a cheap and natural agricultural byproduct (autoclaved maize bran) and its biotransformation into vanillic acid and/or vanillin with a limited number of steps. On the one hand, the potentialities of A. niger I-1472 to produce high levels of polysaccharide-degrading enzymes including feruloyl esterases and to transform ferulic acid into vanillic acid were successfully combined for the release of free ferulic acid from autoclaved maize bran. Then vanillic acid was recovered and efficiently transformed into vanillin by P. cinnabarinus MUCL 39533, since 767 mg/L of biotechnologic vanillin could be produced in the presence of cellobiose and XAD-2 resin. On the other hand, 3-d-old high-density cultures of P. cinnabarinus MUCL39533 could be fed with the autoclaved fraction of maize bran as a ferulic acid source and a. niger I-1472 culture filtrate as an extracellular enzyme source. Under these conditions, P. cinnabarinus MUCL39533 was shown to directly biotransform free ferulic acid released from the autoclaved maize bran by A. niger I-1472 enzymes into 584 mg/L of vanillin. These processes, involving physical, enzymic, and fungal treatments, permitted us to produce crystallin vanillin from autoclaved maize bran without any purification step.

Purification, crystallisation and X-ray diffraction study of fully functional laccases from two ligninolytic fungi

Laccase isozymes from the white-rot basidiomycete fungi Trametes versicolor and Pycnoporus cinnabarinus were purified to apparent iso-electric homogeneity and crystallised. T. versicolor laccase crystallises in two crystal forms, both with the orthorhombic space group P2(1)2(1)2(1), which diffract to 1.9 and 2.95 A resolution, respectively. The crystals of P. cinnabarinus laccase belong to the monoclinic space group C2 and diffract to at least 2.2 A resolution. All the laccase crystals are suitable for X-ray structure determination and contain a full complement of copper ions.