Publications by authors named "Didier Chevret"

32 Publications

Benefits of Iterative Searches of Large Databases to Interpret Large Human Gut Metaproteomic Data Sets.

J Proteome Res 2021 Feb 2. Epub 2021 Feb 2.

Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78350, Jouy-en-Josas, France.

The gut microbiota are increasingly considered as a main partner of human health. Metaproteomics enables us to move from the functional potential revealed by metagenomics to the functions actually operating in the microbiome. However, metaproteome deciphering remains challenging. In particular, confident interpretation of a myriad of MS/MS spectra can only be pursued with smart database searches. Here, we compare the interpretation of MS/MS data sets from 48 individual human gut microbiomes using three interrogation strategies of the dedicated Integrated nonredundant Gene Catalog (IGC 9.9 million genes from 1267 individual fecal samples) together with the database: the classical single-step interrogation strategy and two iterative strategies (in either two or three steps) aimed at preselecting a reduced-sized, more targeted search space for the final peptide spectrum matching. Both iterative searches outperformed the single-step classical search in terms of the number of peptides and protein clusters identified and the depth of taxonomic and functional knowledge, and this was the most convincing with the three-step approach. However, iterative searches do not help in reducing variability of repeated analyses, which is inherent to the traditional data-dependent acquisition mode, but this variability did not affect the hierarchical relationship between replicates and all other samples.
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http://dx.doi.org/10.1021/acs.jproteome.0c00669DOI Listing
February 2021

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

DNA Res 2020 Apr;27(2)

INRAE, UMR1163, Biodiversity and Biotechnology of Fungi, Aix Marseille University, 13009 Marseille, France.

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.
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http://dx.doi.org/10.1093/dnares/dsaa011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406137PMC
April 2020

AA16, a new lytic polysaccharide monooxygenase family identified in fungal secretomes.

Biotechnol Biofuels 2019 16;12:55. Epub 2019 Mar 16.

1Biodiversité et Biotechnologie Fongiques, UMR1163, INRA, Aix Marseille Université, Marseille, France.

Background: Lignocellulosic biomass is considered as a promising alternative to fossil resources for the production of fuels, materials and chemicals. Efficient enzymatic systems are needed to degrade the plant cell wall and overcome its recalcitrance. A widely used producer of cellulolytic cocktails is the ascomycete , but this organism secretes a limited set of enzymes. To improve the saccharification yields, one strategy is to upgrade the enzyme cocktail with enzymes produced by other biomass-degrading filamentous fungi isolated from biodiversity.

Results: In this study, the enzymatic cocktails secreted by five strains from the genus ( strains BRFM 405, 1487, 1489, 1490 and strain BRFM 430) were tested for their ability to boost a reference cocktail for the saccharification of pretreated biomass. Proteomic analysis of fungal secretomes that significantly improved biomass degradation showed that the presence of proteins belonging to a putative LPMO family previously identified by genome analysis and awaiting experimental demonstration of activity. Members of this novel LPMO family, named AA16, are encountered in fungi and oomycetes with life styles oriented toward interactions with plant biomass. One AA16 protein from (AaAA16) was produced to high level in LPMO-type enzyme activity was demonstrated on cellulose with oxidative cleavage at the C1 position of the glucose unit. AaAA16 LPMO was found to significantly improve the activity of CBHI on cellulosic substrates.

Conclusions: Although spp. has been investigated for decades for their CAZymes diversity, we identified members of a new fungal LPMO family using secretomics and functional assays. Properties of the founding member of the AA16 family characterized herein could be of interest for use in biorefineries.
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http://dx.doi.org/10.1186/s13068-019-1394-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420742PMC
March 2019

Mycoplasmas are no exception to extracellular vesicles release: Revisiting old concepts.

PLoS One 2018 28;13(11):e0208160. Epub 2018 Nov 28.

Université de Lyon, Anses, Laboratoire de Lyon, UMR Mycoplasmoses des Ruminants, Lyon, France.

Release of extracellular vesicles (EV) by Gram-negative and positive bacteria is being frequently reported. EV are nano-sized, membrane-derived, non-self-replicating, spherical structures shed into the extracellular environment that could play a role in bacteria-host interactions. Evidence of EV production in bacteria belonging to the class Mollicutes, which are wall-less, is mainly restricted to the genus Acholeplasma and is scanty for the Mycoplasma genus that comprises major human and animal pathogens. Here EV release by six Mycoplasma (sub)species of clinical importance was investigated. EV were obtained under nutritional stress conditions, purified by ultracentrifugation and observed by electron microscopy. The membrane proteins of EV from three different species were further identified by mass spectrometry as a preliminary approach to determining their potential role in host-pathogen interactions. EV were shown to be released by all six (sub)species although their quantities and sizes (30-220 nm) were very variable. EV purification was complicated by the minute size of viable mycoplasmal cells. The proteins of EV-membranes from three (sub)species included major components of host-pathogen interactions, suggesting that EV could contribute to make the host-pathogen interplay more complex. The process behind EV release has yet to be deciphered, although several observations demonstrated their active release from the plasma membrane of living cells. This work shed new light on old concepts of "elementary bodies" and "not-cell bound antigens".
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0208160PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6261642PMC
May 2019

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

Biotechnol Biofuels 2018 2;11:217. Epub 2018 Aug 2.

1UMR1163 BBF Biodiversité et Biotechnologie Fongiques, INRA, Aix Marseille Univ, 13288 Marseille Cedex 09, France.

Background: Lavender () and lavandin (a sterile hybrid of  × ) 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, β-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 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.
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http://dx.doi.org/10.1186/s13068-018-1218-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071384PMC
August 2018

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

Biotechnol Biofuels 2018 23;11:201. Epub 2018 Jul 23.

1Aix Marseille Univ, INRA, UMR 1163, Biodiversité et Biotechnologie Fongiques, BBF, Marseille, France.

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 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 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 , 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.
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http://dx.doi.org/10.1186/s13068-018-1198-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6055342PMC
July 2018

Enzyme Activities of Two Recombinant Heme-Containing Peroxidases, DyP1 and VP2, Identified from the Secretome of Trametes versicolor.

Appl Environ Microbiol 2018 04 2;84(8). Epub 2018 Apr 2.

INRA, Aix-Marseille Université, UMR1163, Biodiversité et Biotechnologie Fongiques, Marseille, France

is a wood-inhabiting agaricomycete known for its ability to cause strong white-rot decay on hardwood and for its high tolerance of phenolic compounds. The goal of the present work was to gain insights into the molecular biology and biochemistry of the heme-including class II and dye-decolorizing peroxidases secreted by this fungus. Proteomic analysis of the secretome of BRFM 1218 grown on oak wood revealed a set of 200 secreted proteins, among which were the dye-decolorizing peroxidase DyP1 and the versatile peroxidase VP2. Both peroxidases were heterologously produced in , biochemically characterized, and tested for the ability to oxidize complex substrates. Both peroxidases were found to be active against several substrates under acidic conditions, and DyP1 was very stable over a relatively large pH range of 2.0 to 6.0, while VP2 was more stable at pH 5.0 to 6.0 only. The thermostability of both enzymes was also tested, and DyP1 was globally found to be more stable than VP2. After 180 min of incubation at temperatures ranging from 30 to 50°C, the activity of VP2 drastically decreased, with 10 to 30% of the initial activity retained. Under the same conditions, DyP1 retained 20 to 80% of its enzyme activity. The two proteins were catalytically characterized, and VP2 was shown to accept a wider range of reducing substrates than DyP1. Furthermore, both enzymes were found to be active against two flavonoids, quercetin and catechin, found in oak wood, with VP2 displaying more rapid oxidation of the two compounds. They were tested for the ability to decolorize five industrial dyes, and VP2 presented a greater ability to oxidize and decolorize the dye substrates than DyP1. is a wood-inhabiting agaricomycete known for its ability to cause strong white-rot decay on hardwood and for its high tolerance of phenolic compounds. Among white-rot fungi, the basidiomycete has been extensively studied for its ability to degrade wood, specifically lignin, thanks to an extracellular oxidative enzymatic system. The corresponding oxidative system was previously studied in several works for classical lignin and manganese peroxidases, and in this study, two new components of the oxidative system of , one dye-decolorizing peroxidase and one versatile peroxidase, were biochemically characterized in depth and compared to other fungal peroxidases.
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http://dx.doi.org/10.1128/AEM.02826-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5881066PMC
April 2018

Profiling of Proteome in Exponential and Stationary Phase of Growth.

Front Microbiol 2017 18;8:913. Epub 2017 May 18.

SECALIM UMR1014, Institut National de la Recherche AgronomiqueNantes, France.

has been reported as a major cause of bacterial food-borne enteritides in developed countries during the last decade. Despite its fastidious growth requirements, including low level of oxygen and high level of CO, this pathogen is able to persist in the environment without permanent loss of its viability and virulence. As is not able to multiply outside a host, the cells spend significant amount of time in stationary phase of growth. The entry into the stationary phase is often correlated to resistance to various stresses in bacteria. The switching between exponential and stationary phases is frequently mediated by the regulator sigma S (RpoS). However, this factor is absent in and molecular mechanisms responsible for transition of cells to the stationary phase remain elusive. In this work, proteomic profiles of cells from exponential and stationary phases were compared using 2-D electrophoresis (2DE) fingerprinting combined with mass spectrometry analysis and qRT-PCR. The identified proteins, whose expression differed between the two phases, are mostly involved in protein biosynthesis, carbon metabolism, stress response and motility. Altered expression was observed also in the pleiotropic regulator CosR that was over-expressed during stationary phase. A shift between transcript and protein level evolution of CosR throughout the growth of was observed using qRT-PCR and (2DE). From these data, we hypothesized that CosR could undergo a negative autoregulation in stationary phase. A consensus sequence resulting from promoter sequence alignment of genes potentially regulated by CosR, including its own upstream region, among strains is proposed. To verify experimentally the potential autoregulation of CosR at the DNA level, electrophoretic mobility shift assay was performed with DNA fragments of CosR promoter region and rCosR. Different migration pattern of the promoter fragments indicates the binding capacity of CosR, suggesting its auto-regulation potential.
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http://dx.doi.org/10.3389/fmicb.2017.00913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5435804PMC
May 2017

The integrative omics of white-rot fungus Pycnoporus coccineus reveals co-regulated CAZymes for orchestrated lignocellulose breakdown.

PLoS One 2017 10;12(4):e0175528. Epub 2017 Apr 10.

Aix-Marseille Université, INRA, UMR 1163, Biodiversité et Biotechnologie Fongiques, BBF, Marseille, France.

Innovative green technologies are of importance for converting plant wastes into renewable sources for materials, chemicals and energy. However, recycling agricultural and forestry wastes is a challenge. A solution may be found in the forest. Saprotrophic white-rot fungi are able to convert dead plants into consumable carbon sources. Specialized fungal enzymes can be utilized for breaking down hard plant biopolymers. Thus, understanding the enzymatic machineries of such fungi gives us hints for the efficient decomposition of plant materials. Using the saprotrophic white-rot fungus Pycnoporus coccineus as a fungal model, we examined the dynamics of transcriptomic and secretomic responses to different types of lignocellulosic substrates at two time points. Our integrative omics pipeline (SHIN+GO) enabled us to compress layers of biological information into simple heatmaps, allowing for visual inspection of the data. We identified co-regulated genes with corresponding co-secreted enzymes, and the biological roles were extrapolated with the enriched Carbohydrate-Active Enzyme (CAZymes) and functional annotations. We observed the fungal early responses for the degradation of lignocellulosic substrates including; 1) simultaneous expression of CAZy genes and secretion of the enzymes acting on diverse glycosidic bonds in cellulose, hemicelluloses and their side chains or lignin (i.e. hydrolases, esterases and oxido-reductases); 2) the key role of lytic polysaccharide monooxygenases (LPMO); 3) the early transcriptional regulation of lignin active peroxidases; 4) the induction of detoxification processes dealing with biomass-derived compounds; and 5) the frequent attachments of the carbohydrate binding module 1 (CBM1) to enzymes from the lignocellulose-responsive genes. Our omics combining methods and related biological findings may contribute to the knowledge of fungal systems biology and facilitate the optimization of fungal enzyme cocktails for various industrial applications.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0175528PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5386290PMC
September 2017

NV Proteins of Fish Novirhabdovirus Recruit Cellular PPM1Bb Protein Phosphatase and Antagonize RIG-I-Mediated IFN Induction.

Sci Rep 2017 03 9;7:44025. Epub 2017 Mar 9.

VIM, INRA, Université Paris-Saclay, 78350, Jouy-en-Josas, France.

Non virion (NV) protein expression is critical for fish Novirhabdovirus, viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV), in vivo pathogenesis. However, the mechanism by which NV promotes the viral replication is still unclear. We developed an approach based on reverse genetics and interactomic and identified several NV-associated cellular partners underlying cellular pathways as potential viral targets. Among these cell partners, we showed that NV proteins specifically interact with a protein phosphatase, Mg/Mn-dependent, 1Bb (PPM1Bb) and recruit it in the close vicinity of mitochondria, a subcellular compartment important for retinoic acid-inducible gene-I- (RIG-I)-mediated interferon induction pathway. PPM1B proteins belong to the PP2C family of serine/threonine (Ser/Thr) protein phosphatase and have recently been shown to negatively regulate the host antiviral response via dephosphorylating Traf family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1). We demonstrated that NV proteins and PPM1Bb counteract RIG-I- and TBK1-dependent interferon (IFN) and IFN-stimulated gene promoter induction in fish cells and, hence, the establishment of an antiviral state. Furthermore, the expression of VHSV NV strongly reduced TBK1 phosphorylation and thus its activation. Our findings provide evidence for a previously undescribed mechanism by which a viral protein recruits PPM1Bb protein phosphatase to subvert innate immune recognition.
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http://dx.doi.org/10.1038/srep44025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5343655PMC
March 2017

Inactivation of Cellobiose Dehydrogenases Modifies the Cellulose Degradation Mechanism of Podospora anserina.

Appl Environ Microbiol 2017 01 30;83(2). Epub 2016 Dec 30.

INRA, Aix Marseille Université, Polytech Marseille, UMR1163 Biodiversité et Biotechnologie Fongiques, Marseille, France

Conversion of biomass into high-value products, including biofuels, is of great interest to developing sustainable biorefineries. Fungi are an inexhaustible source of enzymes to degrade plant biomass. Cellobiose dehydrogenases (CDHs) play an important role in the breakdown through synergistic action with fungal lytic polysaccharide monooxygenases (LPMOs). The three CDH genes of the model fungus Podospora anserina were inactivated, resulting in single and multiple CDH mutants. We detected almost no difference in growth and fertility of the mutants on various lignocellulose sources, except on crystalline cellulose, on which a 2-fold decrease in fertility of the mutants lacking P. anserina CDH1 (PaCDH1) and PaCDH2 was observed. A striking difference between wild-type and mutant secretomes was observed. The secretome of the mutant lacking all CDHs contained five beta-glucosidases, whereas the wild type had only one. P. anserina seems to compensate for the lack of CDH with secretion of beta-glucosidases. The addition of P. anserina LPMO to either the wild-type or mutant secretome resulted in improvement of cellulose degradation in both cases, suggesting that other redox partners present in the mutant secretome provided electrons to LPMOs. Overall, the data showed that oxidative degradation of cellulosic biomass relies on different types of mechanisms in fungi.

Importance: Plant biomass degradation by fungi is a complex process involving dozens of enzymes. The roles of each enzyme or enzyme class are not fully understood, and utilization of a model amenable to genetic analysis should increase the comprehension of how fungi cope with highly recalcitrant biomass. Here, we report that the cellobiose dehydrogenases of the model fungus Podospora anserina enable it to consume crystalline cellulose yet seem to play a minor role on actual substrates, such as wood shavings or miscanthus. Analysis of secreted proteins suggests that Podospora anserina compensates for the lack of cellobiose dehydrogenase by increasing beta-glucosidase expression and using an alternate electron donor for LPMO.
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http://dx.doi.org/10.1128/AEM.02716-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5203631PMC
January 2017

Comparison of Proteomics Profiles of Strain Bf under Microaerobic and Aerobic Conditions.

Front Microbiol 2016 13;7:1596. Epub 2016 Oct 13.

LUNAM Université, Oniris, Université de NantesNantes, France; INRA, UMR 1014 SECALIMNantes, France.

accounts for one of the leading causes of foodborne bacterial enteritis in humans. Despite being considered an obligate microaerobic microorganism, is regularly exposed to oxidative stress. However, its adaptive strategies to survive the atmospheric oxygen level during transmission to humans remain unclear. Recently, the clinical strain Bf was singled out for its unexpected ability to grow under ambient atmosphere. Here, we aimed to understand better the biological mechanisms underlying its atypical aerotolerance trait using two-dimensional protein electrophoresis, gene expression, and enzymatic activities. Forty-seven proteins were identified with a significantly different abundance between cultivation under microaerobic and aerobic conditions. The over-expressed proteins in aerobiosis belonged mainly to the oxidative stress response, enzymes of the tricarboxylic acid cycle, iron uptake, and regulation, and amino acid uptake when compared to microaerobic conditions. The higher abundance of proteins related to oxidative stress was correlated to dramatically higher transcript levels of the corresponding encoding genes in aerobic conditions compared to microaerobic conditions. In addition, a higher catalase-equivalent activity in strain Bf was observed. Despite the restricted catabolic capacities of , this study reveals that strain Bf is equipped to withstand oxidative stress. This ability could contribute to emergence and persistence of particular strains of throughout food processing or macrophage attack during human infection.
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http://dx.doi.org/10.3389/fmicb.2016.01596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5061731PMC
October 2016

Visual Comparative Omics of Fungi for Plant Biomass Deconstruction.

Front Microbiol 2016 24;7:1335. Epub 2016 Aug 24.

Aix-Marseille Université, INRA, UMR1163, Biodiversité et Biotechnologie Fongiques Marseille, France.

Wood-decay fungi contain the cellular mechanisms to decompose such plant cell wall components as cellulose, hemicellulose, and lignin. A multi-omics approach to the comparative analysis of wood-decay fungi gives not only new insights into their strategies for decomposing recalcitrant plant biomass, but also an understanding of how to exploit these mechanisms for biotechnological applications. We have developed an analytical workflow, Applied Biomass Conversion Design for Efficient Fungal Green Technology (ABCDEFGT), to simplify the analysis and interpretation of transcriptomic and secretomic data. ABCDEFGT utilizes self-organizing maps for grouping genes with similar transcription patterns, and an overlay with secreted proteins. The key feature of ABCDEFGT is simple graphic outputs of genome-wide transcriptomic and secretomic topographies, which enables visual inspection without a priori of the omics data and facilitates discoveries of co-regulated genes and proteins. Genome-wide omics landscapes were built with the newly sequenced fungal species Pycnoporus coccineus, Pycnoporus sanguineus, and Pycnoporus cinnabarinus grown on various carbon sources. Integration of the post-genomic data revealed a global overlap, confirming the pertinence of the genome-wide approach. ABCDEFGT was evaluated by comparison with the latest clustering method for ease of output interpretation, and ABCDEFGT gave a better biological representation of fungal behaviors. The genome-wide multi-omics strategy allowed us to determine the potential synergy of particular enzymes decomposing cellulose, hemicellulose, and lignin such as Lytic Polysaccharide Monooxygenases, modular enzymes associated with a cellulose binding module1, and Class II Peroxidase isoforms co-regulated with oxido-reductases. Overall, ABCDEFGT was capable of visualizing genome-wide transcriptional and secretomic profiles for intuitive interpretations and is suitable for exploration of newly-sequenced organisms.
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http://dx.doi.org/10.3389/fmicb.2016.01335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4996036PMC
September 2016

Enhanced degradation of softwood versus hardwood by the white-rot fungus Pycnoporus coccineus.

Biotechnol Biofuels 2015 18;8:216. Epub 2015 Dec 18.

Aix Marseille Université, UMR1163 Biodiversité et Biotechnologie Fongiques, 163 avenue de Luminy, 13288 Marseille, France ; INRA, UMR1163 Biodiversité et Biotechnologie Fongiques, 163 avenue de Luminy, 13288 Marseille, France ; Polytech'Marseille, UMR1163 Biodiversité et Biotechnologie Fongiques, 163 avenue de Luminy, 13288 Marseille, France.

Background: White-rot basidiomycete fungi are potent degraders of plant biomass, with the ability to mineralize all lignocellulose components. Recent comparative genomics studies showed that these fungi use a wide diversity of enzymes for wood degradation. Deeper functional analyses are however necessary to understand the enzymatic mechanisms leading to lignocellulose breakdown. The Polyporale fungus Pycnoporus coccineus BRFM310 grows well on both coniferous and deciduous wood. In the present study, we analyzed the early response of the fungus to softwood (pine) and hardwood (aspen) feedstocks and tested the effect of the secreted enzymes on lignocellulose deconstruction.

Results: Transcriptomic and proteomic analyses revealed that P. coccineus grown separately on pine and aspen displayed similar sets of transcripts and enzymes implicated in lignin and polysaccharide degradation. In particular, the expression of lignin-targeting oxidoreductases, such as manganese peroxidases, increased upon cultivation on both woods. The sets of enzymes secreted during growth on both pine and aspen were more efficient in saccharide release from pine than from aspen, and characterization of the residual solids revealed polysaccharide conversion on both pine and aspen fiber surfaces.

Conclusion: The combined analysis of soluble sugars and solid residues showed the suitability of P. coccineus secreted enzymes for softwood degradation. Analyses of solubilized products and residual surface chemistries of enzyme-treated wood samples pointed to differences in fiber penetration by different P. coccineus secretomes. Accordingly, beyond the variety of CAZymes identified in P. coccineus genome, transcriptome and secretome, we discuss several parameters such as the abundance of manganese peroxidases and the potential role of cytochrome P450s and pectin degradation on the efficacy of fungi for softwood conversion.
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http://dx.doi.org/10.1186/s13068-015-0407-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4683735PMC
December 2015

Shotgun metaproteomic profiling of biomimetic anaerobic digestion processes treating sewage sludge.

Proteomics 2015 Oct 28;15(20):3532-43. Epub 2015 Sep 28.

Irstea, UR HBAN, Antony, France.

Two parallel anaerobic digestion lines were designed to match a "bovid-like" digestive structure. Each of the lines consisted of two continuous stirred tank reactors placed in series and separated by an acidic treatment step. The first line was inoculated with industrial inocula whereas the second was seeded with cow digestive tract contents. After 3 months of continuous sewage sludge feeding, samples were recovered for shotgun metaproteomic and DNA-based analysis. Strikingly, protein-inferred and 16S ribosomal DNA tags based taxonomic community profiles were not consistent. PCA however revealed a similar clustering pattern of the samples, suggesting that reproducible methodological and/or biological factors underlie this observation. The performances of the two digestion lines did not differ significantly and the cow-derived inocula did not establish in the reactors. A low throughput metagenomic dataset (3.4 × 10(6) reads, 1.1 Gb) was also generated for one of the samples. It allowed a substantial increase of the analysis depth (11 vs. 4% of spectral identification rate for the combined samples). Surprisingly, a high proportion of proteins from members of the "Candidatus Competibacter" group, a key microbial player usually found in activated sludge plants, was retrieved in our anaerobic digester samples. Data are available via ProteomeXchange with identifier PXD002420 (http://proteomecentral.proteomexchange.org/dataset/PXD002420).
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http://dx.doi.org/10.1002/pmic.201500041DOI Listing
October 2015

Production, secretion and purification of a correctly folded staphylococcal antigen in Lactococcus lactis.

Microb Cell Fact 2015 Jul 16;14:104. Epub 2015 Jul 16.

INRA, UMR1319 Micalis (Microbiologie de l'Alimentation au service de la Santé), Domaine de Vilvert, 78352, Jouy-en-Josas Cedex, France.

Background: Lactococcus lactis, a lactic acid bacterium traditionally used to ferment milk and manufacture cheeses, is also, in the biotechnology field, an interesting host to produce proteins of medical interest, as it is "Generally Recognized As Safe". Furthermore, as L. lactis naturally secretes only one major endogenous protein (Usp45), the secretion of heterologous proteins in this species facilitates their purification from a protein-poor culture medium. Here, we developed and optimized protein production and secretion in L. lactis to obtain proteins of high quality, both correctly folded and pure to a high extent. As proteins to be produced, we chose the two transmembrane members of the HtrA protease family in Staphylococcus aureus, an important extra-cellular pathogen, as these putative surface-exposed antigens could constitute good targets for vaccine development.

Results: A recombinant ORF encoding a C-terminal, soluble, proteolytically inactive and tagged form of each staphylococcal HtrA protein was cloned into a lactococcal expression-secretion vector. After growth and induction of recombinant gene expression, L. lactis was able to produce and secrete each recombinant rHtrA protein as a stable form that accumulated in the culture medium in similar amounts as the naturally secreted endogenous protein, Usp45. L. lactis growth in fermenters, in particular in a rich optimized medium, led to higher yields for each rHtrA protein. Protein purification from the lactococcal culture medium was easily achieved in one step and allowed recovery of highly pure and stable proteins whose identity was confirmed by mass spectrometry. Although rHtrA proteins were monomeric, they displayed the same secondary structure content, thermal stability and chaperone activity as many other HtrA family members, indicating that they were correctly folded. rHtrA protein immunogenicity was established in mice. The raised polyclonal antibodies allowed studying the expression and subcellular localization of wild type proteins in S. aureus: although both proteins were expressed, only HtrA1 was found to be, as predicted, exposed at the staphylococcal cell surface suggesting that it could be a better candidate for vaccine development.

Conclusions: In this study, an efficient process was developed to produce and secrete putative staphylococcal surface antigens in L. lactis and to purify them to homogeneity in one step from the culture supernatant. This allowed recovering fully folded, stable and pure proteins which constitute promising vaccine candidates to be tested for protection against staphylococcal infection. L. lactis thus proved to be an efficient and competitive cell factory to produce proteins of high quality for medical applications.
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http://dx.doi.org/10.1186/s12934-015-0271-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4502909PMC
July 2015

Interactions between genotype and environment drive the metabolic phenotype within Escherichia coli isolates.

Environ Microbiol 2016 Jan 11;18(1):100-17. Epub 2015 May 11.

Univ Paris-Sud, UMR de Génétique Végétale INRA/Univ Paris-Sud/CNRS, Ferme du Moulon, 91190, Gif-sur-Yvette, France.

To gain insights into the adaptation of the Escherichia coli species to different environments, we monitored protein abundances using quantitative proteomics and measurements of enzymatic activities of central metabolism in a set of five representative strains grown in four contrasted culture media including human urine. Two hundred and thirty seven proteins representative of the genome-scale metabolic network were identified and classified into pathway categories. We found that nutrient resources shape the general orientation of metabolism through coordinated changes in the average abundances of proteins and in enzymatic activities that all belong to the same pathway category. For example, each culture medium induces a specific oxidative response whatever the strain. On the contrary, differences between strains concern isolated proteins and enzymes within pathway categories in single environments. Our study confirms the predominance of genotype by environment interactions at the proteomic and enzyme activity levels. The buffering of genetic variation when considering life-history traits suggests a multiplicity of evolutionary strategies. For instance, the uropathogenic isolate CFT073 shows a deregulation of iron demand and increased oxidative stress response.
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http://dx.doi.org/10.1111/1462-2920.12855DOI Listing
January 2016

Proteome changes during yeast-like and pseudohyphal growth in the biofilm-forming yeast Pichia fermentans.

Amino Acids 2015 Jun 6;47(6):1091-106. Epub 2015 Mar 6.

CNR-IPSP, Consiglio Nazionale delle Ricerche-Dipartimento di Scienze Bio-Agroalimentari, Istituto per la Protezione Sostenibile delle Piante, Area della Ricerca CNR, Via Madonna del Piano 10, 50019, Sesto Fiorentino, Florence, Italy,

The Pichia fermentans strain DISAABA 726 is a biofilm-forming yeast that has been proposed as biocontrol agent to control brown rot on apple. How ever, when inoculated on peach, strain 726 shows yeast-like to pseudohyphal transition coupled to a pathogenic behaviour. To identify the proteins potentially involved in such transition process, a comparative proteome analysis of P. fermentans 726 developed on peach (filamentous growth) vs apple (yeast-like growth) was carried out using two-dimensional gel electrophoresis coupled with mass spectrometry analysis. The proteome comparison was also performed between the two different cell morphologies induced in a liquid medium amended with urea (yeast-like cells) or methionine (filamentous cells) to exclude fruit tissue impact on the transition. Seventy-three protein spots showed significant variations in abundance (±twofold, p < 0.01, confidence intervals 99 %) between pseudohyphal vs yeast-like morphology produced on fruits. Among them, 30 proteins changed their levels when the two morphologies were developed in liquid medium. The identified proteins belong to several pathways and functions, such as glycolysis, amino acid synthesis, chaperones, and signalling transduction. The possible role of a group of proteins belonging to the carbohydrate pathway in the metabolic re-organisation during P. fermentans dimorphic transition is discussed.
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http://dx.doi.org/10.1007/s00726-015-1933-1DOI Listing
June 2015

Fine mapping and characterization of the L-polymerase-binding domain of the respiratory syncytial virus phosphoprotein.

J Virol 2015 Apr 4;89(8):4421-33. Epub 2015 Feb 4.

Unité de Virologie et Immunologie Moléculaires (UR892), INRA, Jouy-en-Josas, France

Unlabelled: The minimum requirement for an active RNA-dependent RNA polymerase of respiratory syncytial virus (RSV) is a complex made of two viral proteins, the polymerase large protein (L) and the phosphoprotein (P). Here we have investigated the domain on P that is responsible for this critical P-L interaction. By use of recombinant proteins and serial deletions, an L binding site was mapped in the C-terminal region of P, just upstream of the N-RNA binding site. The role of this molecular recognition element of about 30 amino acid residues in the L-P interaction and RNA polymerase activity was evaluated in cellula using an RSV minigenome system and site-directed mutagenesis. The results highlighted the critical role of hydrophobic residues located in this region.

Importance: Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine and no good antivirals against RSV are available, it is essential to better understand how the viral machinery functions in order to develop new antiviral strategies. Like all negative-strand RNA viruses, RSV codes for its own machinery to replicate and transcribe its genome. The core of this machinery is composed of two proteins, the phosphoprotein (P) and the large protein (L). Here, using recombinant proteins, we have mapped and characterized the P domain responsible for this L-P interaction and the formation of an active L-P complex. These findings extend our understanding of the mechanism of action of RSV RNA polymerase and allow us to define a new target for the development of drugs against RSV.
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http://dx.doi.org/10.1128/JVI.03619-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4442346PMC
April 2015

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

Biotechnol Biofuels 2014 8;7(1):143. Epub 2014 Oct 8.

INRA, UMR1163 Biotechnologie des Champignons Filamenteux, 13288 Marseille, France ; Aix-Marseille Université, Polytech Marseille, UMR1163 Biotechnologie des Champignons Filamenteux, 13288 Marseille, France.

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.
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http://dx.doi.org/10.1186/s13068-014-0143-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4197297PMC
October 2014

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

BMC Genomics 2014 Jun 18;15:486. Epub 2014 Jun 18.

INRA, UMR1163 Biotechnologie des Champignons Filamenteux, Aix-Marseille Université, Polytech Marseille, 163 avenue de Luminy, CP 925, 13288 Marseille Cedex 09, France.

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.
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http://dx.doi.org/10.1186/1471-2164-15-486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4101180PMC
June 2014

Characterization of an experimental vaccine for bovine respiratory syncytial virus.

Clin Vaccine Immunol 2014 Jul 14;21(7):997-1004. Epub 2014 May 14.

INRA, Unité de Virologie et Immunologie Moléculaires, Jouy-en-Josas, France.

Bovine respiratory syncytial virus (BRSV) and human respiratory syncytial virus (HRSV) are major causes of respiratory disease in calves and children, respectively, and are priorities for vaccine development. We previously demonstrated that an experimental vaccine, BRSV-immunostimulating complex (ISCOM), is effective in calves with maternal antibodies. The present study focuses on the antigenic characterization of this vaccine for the design of new-generation subunit vaccines. The results of our study confirmed the presence of membrane glycoprotein (G), fusion glycoprotein (F), and nucleoprotein (N) proteins in the ISCOMs, and this knowledge was extended by the identification of matrix (M), M2-1, phosphoprotein (P), small hydrophobic protein (SH) and of cellular membrane proteins, such as the integrins αVβ1, αVβ3, and α3β1. The quantity of the major protein F was 4- to 5-fold greater than that of N (∼77 μg versus ∼17 μg/calf dose), whereas G, M, M2-1, P, and SH were likely present in smaller amounts. The polymerase (L), M2-2, nonstructural 1 (NS1), and NS2 proteins were not detected, suggesting that they are not essential for protection. Sera from the BRSV-ISCOM-immunized calves contained high titers of IgG antibody specific for F, G, N, and SH. Antibody responses against M and P were not detected; however, this does not exclude their role in protective T-cell responses. The absence of immunopathological effects of the cellular proteins, such as integrins, needs to be further confirmed, and their possible contribution to adjuvant functions requires elucidation. This work suggests that a combination of several surface and internal proteins should be included in subunit RSV vaccines and identifies absent proteins as potential candidates for differentiating infected from vaccinated animals.
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http://dx.doi.org/10.1128/CVI.00162-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4097437PMC
July 2014

Comparative analyses of Podospora anserina secretomes reveal a large array of lignocellulose-active enzymes.

Appl Microbiol Biotechnol 2014 Sep 3;98(17):7457-69. Epub 2014 Apr 3.

Biotechnologie des Champignons Filamenteux, BCF, UMR 1163, INRA, Aix Marseille Université, Polytech, CP 925, 13228, Marseille Cedex 09, France.

The genome of the coprophilous fungus Podospora anserina harbors a large and highly diverse set of putative lignocellulose-acting enzymes. In this study, we investigated the enzymatic diversity of a broad range of P. anserina secretomes induced by various carbon sources (dextrin, glucose, xylose, arabinose, lactose, cellobiose, saccharose, Avicel, Solka-floc, birchwood xylan, wheat straw, maize bran, and sugar beet pulp (SBP)). Compared with the Trichoderma reesei enzymatic cocktail, P. anserina secretomes displayed similar cellulase, xylanase, and pectinase activities and greater arabinofuranosidase, arabinanase, and galactanase activities. The secretomes were further tested for their capacity to supplement a T. reesei cocktail. Four of them improved significantly the saccharification yield of steam-exploded wheat straw up to 48 %. Fine analysis of the P. anserina secretomes produced with Avicel and SBP using proteomics revealed a large array of CAZymes with a high number of GH6 and GH7 cellulases, CE1 esterases, GH43 arabinofuranosidases, and AA1 laccase-like multicopper oxidases. Moreover, a preponderance of AA9 (formerly GH61) was exclusively produced in the SBP condition. This study brings additional insights into the P. anserina enzymatic machinery and will facilitate the selection of promising targets for the development of future biorefineries.
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http://dx.doi.org/10.1007/s00253-014-5698-3DOI Listing
September 2014

Characterization of salt-adapted secreted lignocellulolytic enzymes from the mangrove fungus Pestalotiopsis sp.

Nat Commun 2013 ;4:1810

INRA, UMR1163-Biotechnologie des Champignons Filamenteux, ESIL-Polytech, Aix-Marseille University, 13288 Marseille, France.

Fungi are important for biomass degradation processes in mangrove forests. Given the presence of sea water in these ecosystems, mangrove fungi are adapted to high salinity. Here we isolate Pestalotiopsis sp. NCi6, a halotolerant and lignocellulolytic mangrove fungus of the order Xylariales. We study its lignocellulolytic enzymes and analyse the effects of salinity on its secretomes. De novo transcriptome sequencing and assembly indicate that this fungus possesses of over 400 putative lignocellulolytic enzymes, including a large fraction involved in lignin degradation. Proteomic analyses of the secretomes suggest that the presence of salt modifies lignocellulolytic enzyme composition, with an increase in the secretion of xylanases and cellulases and a decrease in the production of oxidases. As a result, cellulose and hemicellulose hydrolysis is enhanced but lignin breakdown is reduced. This study highlights the adaptation to salt of mangrove fungi and their potential for biotechnological applications.
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http://dx.doi.org/10.1038/ncomms2850DOI Listing
December 2013

Linking post-translational modifications and variation of phenotypic traits.

Mol Cell Proteomics 2013 Mar 27;12(3):720-35. Epub 2012 Dec 27.

CNRS, UMR 0320/UMR 8120 Génétique Végétale, Gif-sur-Yvette, France.

Enzymes can be post-translationally modified, leading to isoforms with different properties. The phenotypic consequences of the quantitative variability of isoforms have never been studied. We used quantitative proteomics to dissect the relationships between the abundances of the enzymes and isoforms of alcoholic fermentation, metabolic traits, and growth-related traits in Saccharomyces cerevisiae. Although the enzymatic pool allocated to the fermentation proteome was constant over the culture media and the strains considered, there was variation in abundance of individual enzymes and sometimes much more of their isoforms, which suggests the existence of selective constraints on total protein abundance and trade-offs between isoforms. Variations in abundance of some isoforms were significantly associated to metabolic traits and growth-related traits. In particular, cell size and maximum population size were highly correlated to the degree of N-terminal acetylation of the alcohol dehydrogenase. The fermentation proteome was found to be shaped by human selection, through the differential targeting of a few isoforms for each food-processing origin of strains. These results highlight the importance of post-translational modifications in the diversity of metabolic and life-history traits.
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http://dx.doi.org/10.1074/mcp.M112.024349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3591664PMC
March 2013

Polynucleotide phosphorylase has an impact on cell biology of Campylobacter jejuni.

Front Cell Infect Microbiol 2012 14;2:30. Epub 2012 Mar 14.

LUNAM Université, Oniris, University of Nantes Nantes, France.

Polynucleotide phosphorylase (PNPase), encoded by the pnp gene, is known to degrade mRNA, mediating post-transcriptional regulation and may affect cellular functions. The role of PNPase is pleiotropic. As orthologs of the two major ribonucleases (RNase E and RNase II) of Escherichia coli are missing in the Campylobacter jejuni genome, in the current study the focus has been on the C. jejuni ortholog of PNPase. The effect of PNPase mutation on C. jejuni phenotypes and proteome was investigated. The inactivation of the pnp gene reduced significantly the ability of C. jejuni to adhere and to invade Ht-29 cells. Moreover, the pnp mutant strain exhibited a decrease in C. jejuni swimming ability and chick colonization. To explain effects of PNPase on C. jejuni 81-176 phenotype, the proteome of the pnp mutant and parental strains were compared. Overall, little variation in protein production was observed. Despite the predicted role of PNPase in mRNA regulation, the pnp mutation did not induce profound proteomic changes suggesting that other ribonucleases in C. jejuni might ensure this biological function in the absence of PNPase. Nevertheless, synthesis of proteins which are involved in virulence (LuxS, PEB3), motility (N-acetylneuraminic acid synthetase), stress-response (KatA, DnaK, Hsp90), and translation system (EF-Tu, EF-G) were modified in the pnp mutant strain suggesting a more specific role of PNPase in C. jejuni. In conclusion, PNPase deficiency induces limited but important consequences on C. jejuni biology that could explain swimming limitation, chick colonization delay, and the decrease of cell adhesion/invasion ability.
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http://dx.doi.org/10.3389/fcimb.2012.00030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3417634PMC
December 2013

Production and characterization of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) expressed in the oleaginous yeast Yarrowia lipolytica.

Appl Microbiol Biotechnol 2012 Oct 25;96(1):89-101. Epub 2012 May 25.

INRA, UMR1319 Micalis, Domaine de Vilvert, 78350 Jouy-en-Josas, France.

Since its isolation, the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) has been proposed as a new class of therapeutic biological products in the treatment of various diseases. However, the toxicity of this cytokine towards its expression host constitutes a major obstacle to bioprocess development for large-scale production. In this work, the optimized gene encoding hGM-CSF was expressed in the yeast Yarrowia lipolytica in one and two copies under the control of the fatty acid-inducible POX2 promoter. Protein secretion was directed by the targeting sequence of the extracellular lipase (LIP2): preXALip2. After 48 h of induction, Western blot analysis revealed the presence of a nonglycosylated form of 14.5 kDa and a trail of hGM-CSF hyperglycosylated varying from 23 kDa to more than 60 kDa. The two-copy transformants produced hGM-CSF level which was sevenfold higher compared to the single-copy ones. Deglycosylation with PNGase F showed two forms: a mature form of 14.5 kDa and an unprocessed form of 18 kDa. The addition of two alanines to the signal sequence resulted in correct hGM-CSF processing. The production level was estimated at 250 mg/l after preliminary optimization studies of the cultivation and induction phases. The purified hGM-CSF was identified by N-terminal sequencing and LC-MS/MS analysis; its biological activity was confirmed by stimulating the proliferation of TF1 cell line. This study demonstrated that Y. lipolytica is a promising host for the efficient production of active toxic proteins like hGM-CSF.
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http://dx.doi.org/10.1007/s00253-012-4141-xDOI Listing
October 2012

Fusarium verticillioides secretome as a source of auxiliary enzymes to enhance saccharification of wheat straw.

Bioresour Technol 2012 Jun 10;114:589-96. Epub 2012 Mar 10.

INRA, UMR 1163 Biotechnologie des Champignons Filamenteux, 13288 Marseille, France.

Fusarium verticillioides secretes enzymes (secretome), some of which might be potentially useful for saccharification of lignocellulosic biomass since supplementation of commercial cellulases from Trichoderma reesei with the F. verticillioides secretome improved the enzymatic release of glucose, xylose and arabinose from wheat straw by 24%, 88% and 68%, respectively. Determination of enzymatic activities revealed a broad range of hemicellulases and pectinases poorly represented in commercial cocktails. Proteomics approaches identified 57 proteins potentially involved in lignocellulose breakdown among a total of 166 secreted proteins. This analysis highlighted the presence of carbohydrate-active enzymes (CAZymes) targeting pectin (from glycoside hydrolase families GH5, GH27, GH28, GH43, GH51, GH54, GH62, GH88 and GH93, polysaccharide lyase family PL4 and carbohydrate esterase family CE8) and hemicelluloses (from glycoside hydrolase families GH3, GH10, GH11, GH30, GH39, GH43 and GH67). These data provide a first step towards the identification of candidates to supplement T. reesei enzyme preparations for lignocellulose hydrolysis.
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http://dx.doi.org/10.1016/j.biortech.2012.03.009DOI Listing
June 2012

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

BMC Genomics 2012 Feb 2;13:57. Epub 2012 Feb 2.

INRA, UMR BCF, Marseille, France.

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.
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http://dx.doi.org/10.1186/1471-2164-13-57DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3298532PMC
February 2012

Carbohydrate metabolism is essential for the colonization of Streptococcus thermophilus in the digestive tract of gnotobiotic rats.

PLoS One 2011 22;6(12):e28789. Epub 2011 Dec 22.

Commensal and Probiotics-Host Interactions Laboratory, INRA, UMR1319 Micalis, Jouy-en-Josas, France.

Streptococcus thermophilus is the archetype of lactose-adapted bacterium and so far, its sugar metabolism has been mainly investigated in vitro. The objective of this work was to study the impact of lactose and lactose permease on S. thermophilus physiology in the gastrointestinal tract (GIT) of gnotobiotic rats. We used rats mono-associated with LMD-9 strain and receiving 4.5% lactose. This model allowed the analysis of colonization curves of LMD-9, its metabolic profile, its production of lactate and its interaction with the colon epithelium. Lactose induced a rapid and high level of S. thermophilus in the GIT, where its activity led to 49 mM of intra-luminal L-lactate that was related to the induction of mono-carboxylic transporter mRNAs (SLC16A1 and SLC5A8) and p27(Kip1) cell cycle arrest protein in epithelial cells. In the presence of a continuous lactose supply, S. thermophilus recruited proteins involved in glycolysis and induced the metabolism of alternative sugars as sucrose, galactose, and glycogen. Moreover, inactivation of the lactose transporter, LacS, delayed S. thermophilus colonization. Our results show i/that lactose constitutes a limiting factor for colonization of S. thermophilus, ii/that activation of enzymes involved in carbohydrate metabolism constitutes the metabolic signature of S. thermophilus in the GIT, iii/that the production of lactate settles the dialogue with colon epithelium. We propose a metabolic model of management of carbohydrate resources by S. thermophilus in the GIT. Our results are in accord with the rationale that nutritional allegation via consumption of yogurt alleviates the symptoms of lactose intolerance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0028789PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3245227PMC
May 2012