Camille Filiatrault-Chastel
email: camille.filiatrault.chastel@usherbrooke.ca

Senta Heiss-Blanquet
email: senta.blanquet@ifpen.fr

Antoine Margeot
email: antoine.margeot@ifpen.fr

Jean-Guy Berrin
email: jean-guy.berrin@inrae.fr

Camille Filiatrault

Senta Heiss

From fungal secretomes to enzymes cocktails: the path forward to bioeconomy

Keywords: Filamentous fungi, secretome, proteomics, enzymes, CAZymes, cocktail, biorefinery, bioeconomy

Bioeconomy is seen as a way to mitigate the carbon footprint of human activities by reducing at least part of the fossil resources-based economy. In this new paradigm of sustainable development, the use of enzymes as biocatalysts will play an increasing role to provide services and goods. In industry, most of multicomponent enzyme cocktails are of fungal origin. Filamentous fungi secrete complex enzyme sets called "secretomes" that can be utilized as enzyme cocktails to valorize different types of bioresources. In this review, we highlight recent advances in the study of fungal secretomes using improved computational and experimental secretomics methods, the progress in the understanding of industrially important fungi, and the discovery of new enzymatic mechanisms and interplays to degrade renewable resources rich in polysaccharides (e.g. cellulose). We review current biotechnological applications focusing on the benefits and challenges of fungal secretomes for industrial applications with some examples of commercial cocktails of fungal origin containing carbohydrateactive enzymes (CAZymes) and we discuss future trends.

Highlights

• Filamentous fungi secrete high amounts of various enzymes, called "secretome', to fulfill their exodigester lifestyle • Recent development of bioinformatics and proteomics tools facilitate the characterization of fungal secretomes • The exploration of fungal secretomes allowed the discovery of new enzymatic activities • Fungal secretomes offer flexible and convenient solutions for processing complex substrates (e.g. lignocellulose)

• Fungal secretomes have found applications in several markets

Introduction

Bioeconomy can be defined as an economy that relies on the production and conversion of renewable biological resources to produce food, energy, and goods. Its development is seen as a way to mitigate the carbon footprint of human activities by reducing at least part of the fossil resources-based economy (1), and it could help to meet at least half of the UN Sustainable Development Goals [START_REF] El-Chichakli | Five cornerstones of a global bioeconomy[END_REF]. In this context, the use of biotechnological solutions (micro-organisms and enzymes) is believed to play an increasing role, as they have been tailored by evolution to efficiently degrade and modify biopolymers and synthesize biological molecules.

The global enzyme market was already valued at ~10 billion US $ in 2019 and is believed to keep growing at a rate between 5 and 7% per year [START_REF]Enzymes Market Size & Share Industry Report[END_REF]. Among these commercial enzymes, carbohydrate-active enzymes (CAZymes) have the highest market share, before proteases and others, and their global market is forecasted to generate 5.4 billion US $ revenue in 2024 (4). In industry, most of the enzyme cocktails used in so-called "white" biotechnologies are of fungal origin [START_REF] Mckelvey | Biotechnological Use of Fungal Enzymes[END_REF], with a wide range of applications (textiles, food & beverages, animal feed, pulp & paper, cosmetics, nutraceuticals & pharmaceuticals, household care, bioenergy, etc.).

In nature, filamentous fungi are heterotrophs and exodigesters, feeding on organic matter. Fungal life and growth hence rely on the extracellular secretion of various proteins involved in nutrients acquisition, sensing and signaling, cell walls building and remodeling, and competition with other organisms. The entire set of proteins secreted under given circumstances by an organism is called a "secretome", the broad sense of this term includes both freely released proteins and proteins attached to the outer cell wall [START_REF] Tjalsma | Signal Peptide-Dependent Protein Transport in Bacillus subtilis: a Genome-Based Survey of the Secretome[END_REF][START_REF] Girard | Secretomes: The fungal strike force[END_REF]. Using multiple regulation and secretion mechanisms, filamentous fungi can modify their secretome and thus their metabolism in response to changes in their environmental conditions, such as a variation of the available carbon and nitrogen sources [START_REF] Mccotter | Regulation of the fungal secretome[END_REF][START_REF] Brown | How nutritional status signalling coordinates metabolism and lignocellulolytic enzyme secretion[END_REF]. Given that fungi are present in almost all existing ecosystems and have evolved to acquire a broad array of nutritional lifestyles, the nature of these nutrient sources can be remarkably diverse.

Fungi can indeed live as saprophytes, feeding on various types of dead organic material, and/or engage in symbiotic or parasitic relationships with plants, insects, animals. These different lifestyles and nutritional strategies are associated to a wide variety of enzymes specialized in the degradation of complex biopolymers encountered in the environment [START_REF] Krijger | Compositions of fungal secretomes indicate a greater impact of phylogenetic history than lifestyle adaptation[END_REF][START_REF] Lowe | Indifferent, Affectionate, or Deceitful: Lifestyles and Secretomes of Fungi[END_REF]. Some fungal species, although they thrive in similar environments, use different degradation strategies. For instance, fungal wood decayers have been classified empirically according to the different components of the lignocellulosic substrates they target in nature [START_REF] Blanchette | A review of microbial deterioration found in archaeological wood from different environments[END_REF][START_REF] Cragg | Lignocellulose degradation mechanisms across the Tree of Life[END_REF][START_REF] Spatafora | The Fungal Tree of Life: from Molecular Systematics to Genome-Scale Phylogenies[END_REF]: white-rot basidiomycetes degrade the lignin moiety extensively using laccases and peroxidases before attacking cellulose, while brown-rot basidiomycetes induce limited lignin modifications that allow them to gain access to cellulose and hemicellulose using both enzymatic and non-enzymatic reactions, and soft-rot ascomycetes primarily attack plant fibres using cellulases and hemicellulases while leaving a high lignin content. Several genomic studies allowed to link these different strategies to distinct enzyme sets and associated genes, and some of them provided additional details by showing how evolution shaped fungal enzymatic arsenals in regards to their adaptation to diverse habitats and lifestyles [START_REF] Floudas | The Paleozoic Origin of Enzymatic Lignin Decomposition Reconstructed from 31 Fungal Genomes[END_REF][START_REF] Hage | Evolution of Fungal Carbohydrate-Active Enzyme Portfolios and Adaptation to Plant Cell-Wall Polymers[END_REF].

Among the various natural molecules that can be degraded or modified by fungal enzymes, plant-based materials are of particular interest since they are an important reservoir of organic carbon. Lignocellulose in particular is envisioned as a renewable feedstock for the production of biofuels and bioproducts, in order to replace most of the molecules obtained from oil refining that are currently used for the manufacture of consumer products in the fields of health, food, feed, materials, chemicals etc. [START_REF] Cherubini | The biorefinery concept: Using biomass instead of oil for producing energy and chemicals[END_REF][START_REF] Bhowmick | Lignocellulosic biorefinery as a model for sustainable development of biofuels and value added products[END_REF]. One of the main obstacles to the use of this raw material in biorefineries is its recalcitrance, due to the organisation of its constitutive polymers, where crystalline cellulose fibers are surrounded by hemicelluloses and pectin chains and cemented by a lignin matrix [START_REF] Bhowmick | Lignocellulosic biorefinery as a model for sustainable development of biofuels and value added products[END_REF]. An effective way to get access to and transform these polymers is thus to use the natural toolbox provided by saprophytic fungi that have evolved to use plant biomass as a carbon and energy source, and secrete an arsenal of glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, oxidoreductases and hydrogen peroxide-producing enzymes in their secretomes [START_REF] Bouws | Fungal secretomes-nature's toolbox for white biotechnology[END_REF]. By providing a view of the enzymes secreted by fungal strains grown on various substrates, fungal secretomics is thus a powerful tool to better understand biomass degradation mechanisms and design more efficient biomassdegrading enzyme cocktails.

In this review, we highlight recent advances in the study of fungal secretomes using improved computational and experimental secretomics methods, the progress in the understanding of industrially important fungi, and the discovery of new enzymatic mechanisms and interplays involved in the degradation of renewable resources rich in polysaccharides. We review current biotechnological applications focusing on the benefits and challenges of fungal secretomes for industrial applications with some examples of commercial cocktails of fungal origin containing carbohydrate-active enzymes (CAZymes) and we discuss future trends.

Recent advances in the study of fungal secretomes

Improved computational and experimental secretomics methods

The identification of fungal secreted proteins is mainly based on a comparison with existing genomic data, and thus depend on the availability of sequenced genomes for the corresponding organisms. In the last decade, the access to faster and cheaper sequencing methods has facilitated the emergence of large-scale genomics initiatives aiming at the exploration of the fungal kingdom in the frame of health, energy, and environmental issues, thus increasing the number of available fungal genomes. The MycoCosm portal of the US Department of Energy (DOE) Joint Genome Institute (JGI), which counted less than 100 genomes when it was created in 2010 [START_REF] Grigoriev | MycoCosm portal: gearing up for 1000 fungal genomes[END_REF], now includes more than 2000 fungal genomes (https://mycocosm.jgi.doe.gov/, May 2021).

Based on these available genomes, and on the use of algorithms that can identify signal peptides and other characteristic features of secreted proteins, several in silico secretomic databases such as the Fungal Secretome Database [START_REF] Choi | Fungal Secretome Database: Integrated platform for annotation of fungal secretomes[END_REF] and the Fungal Secretome KnowledgeBase [START_REF] Lum | FunSecKB: the Fungal Secretome KnowledgeBase[END_REF][START_REF] Meinken | FunSecKB2: a fungal protein subcellular location knowledgebase[END_REF] have been created. Although some care must be taken with omissions and secretion prediction errors, these tools can be used to predict a catalog of all the proteins that a given organism can secrete. In addition to these bioinformatic methods, experimental secretomics is needed to account for the response to specific environmental conditions. It is indeed a powerful tool that allows to compare protein secretion profiles during growth on different substrates, to study the dynamics of protein secretion and to identify co-secretion patterns. In this regard, it could be useful to establish dedicated databases gathering the composition of fungal secretomes under varying conditions, with a link to existing protein databases for expert annotation of protein functions, such as CAZy for carbohydrate-active enzymes (www.cazy.org) [START_REF] Lombard | The carbohydrate-active enzymes database (CAZy) in 2013[END_REF], Merops for proteases (www.ebi.ac.uk/merops/) [START_REF] Rawlings | The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database[END_REF], etc. The main limitation is the lack of homogeneity between datasets due to discrepancies between the methods used to grow microorganisms, collect and analyze the secretomes.

Preparation of fungal secretome samples is usually done by growing fungal strains on chosen soluble or insoluble substrates in liquid cultures, followed by separation of the mycelium from medium containing the secreted proteins and concentration using ultrafiltration or other techniques [START_REF] Vincent | Hunting down fungal secretomes using liquid-phase IEF prior to high resolution 2-DE[END_REF]. To obtain reliable results, it is important to prevent cell lysis, which would contaminate the samples with intracellular proteins and release proteases that could degrade proteins of interest. Another challenge is the binding of some secreted proteins to the cells or to the substrates, especially when substrates are insoluble. Methods can be employed to solubilize this protein fraction [START_REF] Bengtsson | A novel proteomics sample preparation method for secretome analysis of Hypocrea jecorina growing on insoluble substrates[END_REF][START_REF] Tuveng | Proteomic Detection of Carbohydrate-Active Enzymes (CAZymes) in Microbial Secretomes[END_REF], but care is needed because harsh procedures can lead to cell lysis and/or damage protein samples.

Regarding the separation and analysis of the complex proteins mixtures thus prepared, several advances allowed to improve the quality of secretomics studies. Although gel-based techniques such as two-dimensional gel electrophoresis (2-DE) are still used for the separation of proteins, these approaches are time-consuming and can have low resolution and reproducibility limitations [START_REF] Girard | Secretomes: The fungal strike force[END_REF][START_REF] Alfaro | Comparative analysis of secretomes in basidiomycete fungi[END_REF]. Of note, short migration onto 1D SDS-PAGE can remove efficiently impurities (pigments) to facilitate protein digestion and mass spectrometry analysis. Nevertheless, gel-free approaches using liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) seem to be the most widely used. The most common technique is shotgun proteomics (Figure 1 : Principle of shotgun proteomics using LC-MS/MS Figure 1), in which peptide mixtures are generated using proteolytic digestion and fractionated using various chromatography techniques; separated peptides are then submitted to tandem mass spectrometry analysis, and protein identification is performed by comparison to sequence databases [START_REF] Bianco | Methodologies and Perspectives of Proteomics Applied to Filamentous Fungi: From Sample Preparation to Secretome Analysis[END_REF][START_REF] Cologna N De M Di | Exploring Trichoderma and Aspergillus secretomes: Proteomics approaches for the identification of enzymes of biotechnological interest[END_REF]. This increased use of shotgun proteomics also favored the development of quantitative proteomics [START_REF] Adav | Fungal Secretome for Biorefinery: Recent Advances in Proteomic Technology[END_REF][START_REF] Otto | Quantitative proteomics in the field of microbiology[END_REF], using label-based techniques such as ICAT (isotope-coded affinity tag) [START_REF] Gygi | Quantitative analysis of complex protein mixtures using isotope-coded affinity tags[END_REF], SILAC (stable isotope labelling by amino acid) [START_REF] Ong | Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics[END_REF], TMT (tandem mass tags) [START_REF] Thompson | Tandem Mass Tags: A Novel Quantification Strategy for Comparative Analysis of Complex Protein Mixtures by MS/MS[END_REF] or iTRAQ (isobaric tags for relative and absolute quantitation) [START_REF] Ross | Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging Reagents[END_REF], and label free techniques based on spectral counting such as emPAI (exponentially modified protein abundance index) [START_REF] Ishihama | Exponentially Modified Protein Abundance Index (emPAI) for Estimation of Absolute Protein Amount in Proteomics by the Number of Sequenced Peptides per Protein[END_REF], NSAF (normalized spectra abundance factor) [START_REF] Zybailov | Statistical Analysis of Membrane Proteome Expression Changes in Saccharomyces cerevisiae[END_REF], or APEX (absolute protein expression) [START_REF] Lu | Absolute protein expression profiling estimates the relative contributions of transcriptional and translational regulation[END_REF].

Advances in the understanding of industrially important fungi

Unsurprisingly, many of the fungal strains that were included in secretomics studies in the recent years are of industrial relevance, with the objective of better understanding the way filamentous fungi perform biomass degradation and the way their enzymes can be used for biotechnological applications. Among the organisms that are investigated for their secretion of biomass-degrading enzymes, the ascomycete Trichoderma reesei (syn. Hypocrea jecorina) is certainly the most famous, and its cellulolytic system has been extensively studied for more than 70 years [START_REF] Bischof | Cellulases and beyond: the first 70 years of the enzyme producer Trichoderma reesei[END_REF]. Since the sequencing of its genome in 2008 [START_REF] Martinez | Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina)[END_REF], many comparative studies allowed to draw up a catalog of the extra-cellular proteins secreted by several T. reesei strains in response to different conditions, such as pH [START_REF] Adav | Proteomic Analysis of pH and Strains Dependent Protein Secretion of Trichoderma reesei[END_REF] and carbon sources [START_REF] Herpoël-Gimbert | Comparative secretome analyses of two Trichoderma reesei RUT-C30 and CL847 hypersecretory strains[END_REF][START_REF] Jun | Enzyme production by filamentous fungi: analysis of the secretome of Trichoderma reesei grown on unconventional carbon source[END_REF][START_REF] Adav | Quantitative Secretomic Analysis of Trichoderma reesei Strains Reveals Enzymatic Composition for Lignocellulosic Biomass Degradation[END_REF][START_REF] Adav | Protein abundance in multiplexed samples (PAMUS) for quantitation of Trichoderma reesei secretome[END_REF][START_REF] Jun | Insights into enzyme secretion by filamentous fungi: Comparative proteome analysis of Trichoderma reesei grown on different carbon sources[END_REF][START_REF] He | Cost-effective lignocellulolytic enzyme production by Trichoderma reesei on a cane molasses medium[END_REF][START_REF] Dos | Comparative metabolism of cellulose, sophorose and glucose in Trichoderma reesei using high-throughput genomic and proteomic analyses[END_REF][START_REF] Da Silva | Secretomic Analysis Reveals Multi-Enzymatic Complexes in Trichoderma reesei Grown in Media Containing Lactose or Galactose[END_REF][START_REF] Borin | Comparative Secretome Analysis of Trichoderma reesei and Aspergillus niger during Growth on Sugarcane Biomass[END_REF][START_REF] Arntzen | Quantitative comparison of the biomass-degrading enzyme repertoires of five filamentous fungi[END_REF] (see examples in Table 1). These studies allowed to better understand the regulation effects exerted by simple sugars commonly used to induce the secretion of proteins (lactose, galactose, xylose, sophorose), as well as by various plant-based types of biomass (cellulose, starch, sugarcane molasses and bagasse, saw dust, corn stover, etc.), thus providing valuable hints on the ideal conditions for the cost-effective production of cellulolytic cocktails.

Species of the genus Aspergillus are also among the most well-known and studied filamentous fungi, several of them being already used for the industrial production of organic acids and enzymes, with applications in food, feed, textile, or pulp and paper industries [START_REF] Park | Diversity, Application, and Synthetic Biology of Industrially Important Aspergillus Fungi[END_REF]. Most of the saprophytic Aspergillus species are able to secrete high quantities of plant cell wall degrading enzymes [START_REF] Vries Rp De | Aspergillus Enzymes Involved in Degradation of Plant Cell Wall Polysaccharides[END_REF][START_REF] Culleton | Physiological and molecular aspects of degradation of plant polysaccharides by fungi: What have we learned from Aspergillus?[END_REF], and several studies have examined the wide range of enzymes produced on various synthetic or complex substrates by A. terreus [START_REF] Arntzen | Quantitative comparison of the biomass-degrading enzyme repertoires of five filamentous fungi[END_REF][START_REF] Han | Extracellular proteome of Aspergillus terreus grown on different carbon sources[END_REF], A. niger [START_REF] Lu | The intra-and extracellular proteome of Aspergillus niger growing on defined medium with xylose or maltose as carbon substrate[END_REF][START_REF] Shi | Physicochemical Properties Analysis and Secretome of Aspergillus niger in Fermented Rapeseed Meal[END_REF], A. oryzae [START_REF] Zhang | Secretome of Aspergillus oryzae in Shaoxing rice wine koji[END_REF], A. fumigatus [START_REF] Sharma | Evaluation of Glycosyl Hydrolases in the Secretome of Aspergillus fumigatus and Saccharification of Alkali-Treated Rice Straw[END_REF][START_REF] Adav | Quantitative proteomic study of Aspergillus fumigatus secretome revealed deamidation of secretory enzymes[END_REF], A. nidulans [START_REF] Nekiunaite | Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by Aspergillus nidulans grown on different starches[END_REF] and A. japonicus [START_REF] Filiatrault-Chastel | AA16, a new lytic polysaccharide monooxygenase family identified in fungal secretomes[END_REF]. Secretomic studies on the main Aspergillus species, as well as five Trichoderma species including T. reesei, were extensively reviewed in 2018 by di Cologna et al. [START_REF] Cologna N De M Di | Exploring Trichoderma and Aspergillus secretomes: Proteomics approaches for the identification of enzymes of biotechnological interest[END_REF].

Beside these industrial workhorses, secretomics approaches also allowed to get a better comprehension of the biology of other well-known ascomycetes such as the plant pathogen Fusarium graminearum [START_REF] Yang | Secretomics identifies Fusarium graminearum proteins involved in the interaction with barley and wheat[END_REF][START_REF] Ji | Shotgun Analysis of the Secretome of Fusarium graminearum[END_REF], the coprophilous ascomycete Podospora anserina [START_REF] Poidevin | Comparative analyses of Podospora anserina secretomes reveal a large array of lignocellulose-active enzymes[END_REF][START_REF] Mäkelä | Cultivation of Podospora anserina on soybean hulls results in an efficient enzyme cocktail for plant biomass hydrolysis[END_REF], the model organism Neurospora crassa [START_REF] Arntzen | Quantitative comparison of the biomass-degrading enzyme repertoires of five filamentous fungi[END_REF][START_REF] Phillips | Quantitative Proteomic Approach for Cellulose Degradation by Neurospora crassa[END_REF][START_REF] Xiong | The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation[END_REF] and others. Increasing attention is given to basidiomycetes such as Phanerochaete chrysosporium or Postia placenta, due to their ability to degrade or modify all the components of woody biomass [START_REF] Alfaro | Comparative analysis of secretomes in basidiomycete fungi[END_REF][START_REF] Rytioja | Plant-Polysaccharide-Degrading Enzymes from Basidiomycetes[END_REF][START_REF] Gauna | Secretome characterization of the lignocellulose-degrading fungi Pycnoporus sanguineus and Ganoderma resinaceum growing on Panicum prionitis biomass[END_REF][START_REF] Machado | The Secretome of Phanerochaete chrysosporium and Trametes versicolor Grown in Microcrystalline Cellulose and Use of the Enzymes for Hydrolysis of Lignocellulosic Materials[END_REF], and recent studies have focused on the investigation of the distinct degradation strategies of white rot, brown rot and litter rot basidiomycetes [START_REF] Presley | Coupling Secretomics with Enzyme Activities To Compare the Temporal Processes of Wood Metabolism among White and Brown Rot Fungi[END_REF][START_REF] Miyauchi | Conserved white-rot enzymatic mechanism for wood decay in the Basidiomycota genus Pycnoporus[END_REF][START_REF] Navarro | Fast solubilization of recalcitrant cellulosic biomass by the basidiomycete fungus Laetisaria arvalis involves successive secretion of oxidative and hydrolytic enzymes[END_REF].

Discovery of new enzymatic mechanisms and interplays

The main fraction of proteins secreted by filamentous fungi upon growth on plant-based carbon sources belong to CAZymes that can degrade or modify polysaccharides such as cellulose, hemicelluloses and starch. Most of these enzymes, which have been the focus of many studies for decades due to their industrial interest, were found to break glycosidic bonds using an hydrolytic mechanism. The established paradigm of polysaccharides breakdown was revisited when the mode of action of two enzyme families (GH61 and CBM33), respectively secreted by fungi and bacteria, was unveiled [START_REF] Harris | Stimulation of Lignocellulosic Biomass Hydrolysis by Proteins of Glycoside Hydrolase Family 61: Structure and Function of a Large, Enigmatic Family[END_REF][START_REF] Vaaje-Kolstad | An Oxidative Enzyme Boosting the Enzymatic Conversion of Recalcitrant Polysaccharides[END_REF]. These mono-copper enzymes were found able to degrade chitin and cellulose through a previously unknown oxidative mechanism [START_REF] Harris | Stimulation of Lignocellulosic Biomass Hydrolysis by Proteins of Glycoside Hydrolase Family 61: Structure and Function of a Large, Enigmatic Family[END_REF][START_REF] Vaaje-Kolstad | An Oxidative Enzyme Boosting the Enzymatic Conversion of Recalcitrant Polysaccharides[END_REF][START_REF] Forsberg | Cleavage of cellulose by a CBM33 protein[END_REF][START_REF] Quinlan | Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components[END_REF], relying on the presence of an oxygen atom provided either by dioxygen or hydrogen peroxide [START_REF] Bissaro | Oxidative cleavage of polysaccharides by monocopper enzymes depends on H2O2[END_REF][START_REF] Hangasky | Reactivity of O2 versus H2O2 with polysaccharide monooxygenases[END_REF], and of an extracellular electrondonating system [START_REF] Frommhagen | Distinct Substrate Specificities and Electron-Donating Systems of Fungal Lytic Polysaccharide Monooxygenases[END_REF]. One of the most prominent features of these new enzymes called lytic polysaccharide monooxygenases (LPMOs) is their ability to boost the degradation of various substrates by hydrolytic enzymes, which led to a very strong interest from the scientific and industrial communities [START_REF] Horn | Novel enzymes for the degradation of cellulose[END_REF][START_REF] Johansen | Discovery and industrial applications of lytic polysaccharide mono-oxygenases[END_REF][START_REF] Dotson | Polypeptides Having Cellulolytic Enhancing Activity And Polynucleotides Encoding Same[END_REF][START_REF] Mcfarland | Compositions And Methods For Enhancing The Degradation Or Conversion Of Cellulose-Containing Material[END_REF].

In 2013, a new class called "auxiliary activities" (AA) was created in the CAZy database, to provide a better coverage of the full extent of the lignocellulose breakdown machinery, and to classify LPMO families [START_REF] Levasseur | Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes[END_REF].

This AA class now contains two ligninolytic enzymes families, seven families of oxidoreductases active on carbohydrates and phenolic compounds, and seven LPMO families. Among the five new LPMO families that were added since 2013, four are secreted by fungal species: the AA11 and AA13 families, active respectively on chitin and starch, were discovered using bioinformatics approaches, thanks to sequence homologies with domains carried by known LPMO families [START_REF] Hemsworth | Discovery and characterization of a new family of lytic polysaccharide monooxygenases[END_REF][START_REF] Vu | A family of starch-active polysaccharide monooxygenases[END_REF]; the AA14 family was identified by transcriptomic and secretomic approaches in the white-rot basidiomycete Pycnoporus coccineus [START_REF] Couturier | Lytic xylan oxidases from wood-decay fungi unlock biomass degradation[END_REF], and the founding member of the AA16 family was identified in Aspergillus secretomes [START_REF] Filiatrault-Chastel | AA16, a new lytic polysaccharide monooxygenase family identified in fungal secretomes[END_REF]. Comparative secretomic approaches also allowed to identify several enzymes that are often co-secreted with LPMOs by fungi and can serve as electron and/or H 2 O 2 donors, such as cellobiose dehydrogenases (CDH) and other oxidative enzymes belonging to families AA3, AA5, AA7 and AA12 [START_REF] Berrin | Fungal secretomics to probe the biological functions of lytic polysaccharide monooxygenases[END_REF][START_REF] Bissaro | Oxidoreductases and Reactive Oxygen Species in Conversion of Lignocellulosic Biomass[END_REF][START_REF] Garajova | Single-domain flavoenzymes trigger lytic polysaccharide monooxygenases for oxidative degradation of cellulose[END_REF][START_REF] Momeni | Discovery of fungal oligosaccharide-oxidising flavo-enzymes with previously unknown substrates, redox-activity profiles and interplay with LPMOs[END_REF][START_REF] Várnai | The Pyrroloquinoline-Quinone-Dependent Pyranose Dehydrogenase from Coprinopsis cinerea Drives Lytic Polysaccharide Monooxygenase Action[END_REF][START_REF] Phillips | Cellobiose Dehydrogenase and a Copper-Dependent Polysaccharide Monooxygenase Potentiate Cellulose Degradation by Neurospora crassa[END_REF]. Fungal secretomics, in combination with genomic data information, is therefore a promising approach to discover new enzymatic functions and enzyme interplays.

Current biotechnological applications

Benefits and challenges of fungal secretomes for industrial applications

In addition to being a major source for enzyme discovery, fungal secretomes can be used as commercial products. There are several possible drivers for such developments. The first one is the cost of downstream processing (DSP), which is already low when working with organisms that secrete enzymes, as there is no need to break-up cells. DSP can be further simplified if advanced purification is not needed for the targeted application. This is also enabled by the high secretory capacity of industrial filamentous fungi workhorses.

Indeed, while strains isolated from the environment are relatively low producers and usually do not perform enough for economic viability, screening programs and strain engineering have led to the selection of mutants with secretion capacities improved by more than ten-fold [START_REF] Montenecourt | Selective Screening Methods for the Isolation of High Yielding Cellulase Mutants of Trichoderma reesei[END_REF]. In combination with optimized media and fermentation processes (high-gravity, fed-batch or chemostats), impressive titers have been reported, the most emblematic being the 100 g/L reported for T. reesei strains (100). To take full advantage of these possibilities, it is required to target a market with lower purity requirements such as feed or bioenergy and ensuring that side-activities are not detrimental or toxic for applications. A relevant example is the Novozym ® 188 enzymatic preparation from clarified Aspergillus fermentation broth, sold for its β-glucosidase activity but which is known to contain other enzymes. Indeed, at least two β-glucosidase isoforms have been described (101) and other proteins account for at least 15% of the secretome (102), which includes xylanases and pectinases (103).

Furthermore, there are many industrial applications where a whole array of activities is needed, mostly when the target substrate is complex. The obvious example that comes to mind is the production of (hemi)-cellulases by fungi such as T. reesei, with most industrial applications found in lignocellulosic biomass degradation for the biorefinery industry (104). Engineered industrial strains of T. reesei, Aspergillus niger or Thermothelomyces thermophila (previously Myceliophthora thermophila or Sporotrichum thermophile) are typically reported to produce secretomes containing between 20 to 80 enzymes (105). Some studies even report up to 230 single proteins (47) in T. reesei secretomes. There is a consensus that an efficient degradation of cellulose by aerobic fungal systems requires at least four cellulolytic activities (reducing-end and non-reducing end cellobiohydrolases, endoglucanases and β-glucosidases), keeping in mind that LPMOs are a great supplement. All these enzymes are naturally produced by the above-mentioned (and several others) fungal species in proportions that allow good degradation kinetics. In this context, separate production of the enzyme components and artificial blending is not an obvious industrial advantage. Moreover, the four canonical enzymatic activities hide the fact that fungi secrete enzymes from different CAZy families with apparently redundant activities. For instance, T. reesei, despite being known for its relatively poor enzymatic diversity, secretes five enzymes identified as endo-β-1,4-glucanases coming from four different families (GH5, GH7, GH12 and GH45); precise differences in substrate specificities between these endoglucanases are still not known today. In addition to cellulose-degrading activities, the T. reesei genome contains about 15 genes coding for characterized hemicellulases (and more than 30 others predicted), as well as accessory proteins such as the two "cellulose-induced proteins" CIP1 and CIP2, and swollenin, which does not display any hydrolytic activity but is thought to be involved in the disruption of crystalline cellulose [START_REF] Martinez | Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina)[END_REF]106,107). In this context, using full secretomes for industrial application can be seen as a display of industrial realism, but also as a tribute to the yet unexplained complexity of Nature. However, several studies have obtained good results using simplified, reconstituted secretomes or minimal enzymatic cocktails, which can reach hydrolysis efficiencies comparable to or better than whole secretomes (108-111).

Production of fungal enzymes cocktails

Enzyme production protocols depend on the fungal species and enzymes involved. Submerged fermentation is the dominant and most scalable production process, that fits the high volumes required for the biofuels or commodity chemicals markets. Generally, production of fungal secretomes must be induced in some way, the most straightforward way being to include the target substrate or one of its degradation products in the medium.

For instance, cellulose can act as an inducer for cellulases production, but soluble substrates such as cellobiose, sophorose or xylose can also play this role (112). More intriguingly, lactose has been a popular substrate, an explanation for this being that the glucose-galactose dimer may be recognized as hemicellulose degradation product. In spite of its efficiency, in order to reduce costs for industrial enzyme production it might be advantageous to partially or completely replace well-known but rather expensive sugars such as lactose by other inducers. For example, in case of on-site cellulose production, lignocellulose products generated in the process, such as softwood pulps, but also sugar beet pulps could be used (113-117). In this context, it is important to keep in mind that different substrate compositions may lead to cocktails with different properties (118), and thus different applications. Glucose often has an adverse effect by triggering carbon catabolite repression, which often results in a preference for growth instead of enzyme production. However, as glucose is a readily available substrate during cellulose hydrolysis, genetic engineering has proven to be successful in creating strains able to secrete high enzyme amounts even in the presence of glucose (119).

Besides usual parameters relevant to monitor fermentation (pH, temperature, nitrogen source, etc.), specific attention must be paid both to the filamentous nature of most strains, which increases viscosity of the fermentation broth, and to their obligate aerobic nature, which makes the O 2 delivery an important parameter (120). This fact has stimulated several studies on the morphology of fungi, small branched hyphae or small pellets being preferred to reduce the viscosity of fermentation broth (121,122).

Examples of commercial cocktail from fungal sources

Many commercial enzyme cocktails are secretomes of one or more selected fungal strains. Table 2 shows some examples of widely used secretome-based industrial enzyme cocktails. The origin and composition of such cocktails are not always easily accessible to the public. Moreover, some enzymatic cocktails developed by industry are commercial products and thus are not readily available to the scientific community. It is also not known if genetic modifications have been performed to enrich the secretomes in one or several enzyme activities. For obtaining the desired activity, it can however be sufficient to perform random mutagenesis and selection (123) and optimize fermentation conditions as previously mentioned. Some commercial cocktails have been analyzed in more details and the following exemplifies the importance of these secretome-based cocktails and their applications.

One of the most important industrial enzyme products are pectinase cocktails. They can be used for different applications, such as fruit juice or wine production, synthesis of prebiotics such as fructooligosaccharides (FOS) or galactooligosaccharides (GOS), but also in the textile industry (124). Each application requires slightly different pectinase activities. For example, apple juice extraction requires enzymes facilitating pressing and thus containing a high rhamnogalacturonase activity, which can be found in many Aspergillus species. In the clarification process, small particles need to be degraded and require cocktails containing, besides pectinase activity, also hemicellulases and cellulases (124). These are provided for example by the Rapidase Lignocellulose hydrolysis for biofuel production is an increasingly important issue. The complex nature and recalcitrance of the feedstocks requires very efficient fungal enzymatic cocktails. T. reesei, M. thermophila, Talaromyces cellulolyticus and Penicillium funiculosum are the most important fungal species used for industrial enzyme production. In the last 10 years, important progress has been made and the efficiency of cocktails has been continuously improved, especially following the discovery of LPMOs. Commercial enzyme cocktails produced by the two main enzyme manufacturers Genencor/Dupont and Novozymes are based on T.

reesei secretomes. The Spezyme ® CP produced by Genencor/Dupont contains more than 80 proteins and is similar to the secretome obtained after growth on corn stover (131). A proteomic analysis of this cocktail evidenced the presence of the main cellobiohydrolases and endoglucanases, but also β-glucosidase, xyloglucanase, β-xylosidase, β-mannanase and endo-β-1,6-galaturonidase activities, among others (131-133).

However, its β-glucosidase activity is very low, which is typical for secretomes from T. reesei strains, and this shortcoming was overcome in the subsequent enzyme cocktails belonging to the Accellerase ® series.

Accellerase ® 1000 released consistently more glucose than Spezyme ® CP from a variety of lignocellulosic biomass that had been subjected to different pretreatments. On the other hand, Spezyme CP was shown to release more xylose from the same substrates (134). Similar improvements of T. reesei enzyme cocktails have been realized in the Cellic Ctec ® enzyme series produced by Novozymes. The study of Sun et al. (132) illustrates the increasing β-glucosidase, cellobiohydrolase and filter paper activities of Cellic Ctec ® -1, -2 and -3, which allow substantial decrease of enzyme loadings while obtaining the same or even better hydrolysis yields. Cellic Ctec ® -2 and -3 also contain additional AA9 LPMO enzymes, which contribute to increased efficiencies. Even if oxidized sugars such as gluconic acids are produced, high ethanol yields can be obtained from wheat straw (> 85% of maximum theoretical yield) compared to the yields achieved by a mixture of Celluclast ® and β-glucosidase SP188 (66% of maximum) (135).

Future trends

Discovery of new enzyme sources

Although many research efforts have already been spent on the development of powerful enzymatic cocktails for a large array of applications, the quest for improved industrial performances is still relevant. In addition, new applications for fungal secretomes also emerge, such as delignification and detoxification of biofuel feedstocks by ligninolytic enzyme cocktails (146), control of algal blooms (147), metal toxicity assessment (148) or hydrocarbon decontamination (149). In this perspective, and in order to support the great diversification of substrates and processes that will come with the development of biorefineries and environmental applications, it is important to keep exploring the huge biodiversity offered by fungal species.

The goal of this search is to identify strains displaying more efficient, more diversified or more specialized secretomes than the organisms that are currently used, with the possibility of finding new enzymatic activities and novel synergies. To this end, research groups are collecting fungal strains in extremely diverse environments such as tropical areas, deserts, high mountains, mangroves, animal faeces, energy crops plantations and many more (150-159), in order to produce secretomes and screen them for target activities.

Recent proteomics studies have revealed the polysaccharide degradation potential of species that were until now unexplored : the marine fungus Paradendryphiella salina displays a large CAZyme repertoire conferring the ability to degrade brown algae polysaccharides (160). The wood-rotting fungus Lentinus crinitus secretes a very diverse cocktail including CAZymes and oxidoreductases, which could find application in lignocellulose degradation (161). The secretomes of Aspergillus terreus, Myceliophthora thermophila and Phanerochaete chrysosporium were compared with those of T. reesei, and N. crassa on several substrates.

These largely unexplored fungal species could in addition to Trichoderma and other Aspergillus species provide future production strains for industrial cocktails [START_REF] Arntzen | Quantitative comparison of the biomass-degrading enzyme repertoires of five filamentous fungi[END_REF]162).

To be used even more efficiently in the future, approaches to explore the biodiversity will need to rely on increasingly robust isolation and activity testing methods (163). Fungal metagenomics or meta-transcriptomics approaches are also attractive but may be hampered by the prominence of bacterial genetic material in samples and the difficulty to assemble full gene sequences using current techniques. Single-cell mass spectrometrybased proteomics could also become a powerful tool for single-cell analyses (164) but to our knowledge they have not been applied to the study of fungal secretomes and the development of enzyme cocktails. The use of high-throughput functional screening can be an alternative. Promises and limits of these approaches have been documented elsewhere, but breakthroughs are to be expected in the coming years with new sequencing techniques (165,166). The emerging techniques of metaproteomics and metasecretomics are also powerful tools that will allow to better understand the processes involved in biomass degradation by mixed microbial communities in complex ecosystems such as soils and plant litter (167-170).

An interesting source of plant-biomass degrading enzymes could be offered by understudied and underexploited organisms such as some basidiomycetes [START_REF] Alfaro | Comparative analysis of secretomes in basidiomycete fungi[END_REF][START_REF] Rytioja | Plant-Polysaccharide-Degrading Enzymes from Basidiomycetes[END_REF], but also anaerobic fungi. These are found notably in the digestive tract of large herbivorous mammals, where they are the primary colonizers of ingested plant materials; their high lignocellulolytic potential, which might be related to their ability to synthesize cellulosome complexes able to accommodate a wide repertoire of CAZymes, makes them appealing targets for future secretomic studies (171-174).

Improved enzymatic cocktails

In addition to the optimization of process conditions, several strategies can be used to obtain improved enzymatic cocktails, including the mixture of several secretomes or the supplementation of an existing preparation with lacking enzymatic activities (175). T. reesei cocktails in particular have been subject of numerous supplementation approaches, using isolated enzymes (176-179), crude fungal secretomes [START_REF] Filiatrault-Chastel | AA16, a new lytic polysaccharide monooxygenase family identified in fungal secretomes[END_REF][START_REF] Poidevin | Comparative analyses of Podospora anserina secretomes reveal a large array of lignocellulose-active enzymes[END_REF][START_REF] Navarro | Fast solubilization of recalcitrant cellulosic biomass by the basidiomycete fungus Laetisaria arvalis involves successive secretion of oxidative and hydrolytic enzymes[END_REF]103,151,(180)(181)(182)(183)(184)(185)(186) or commercial enzymatic preparations (187-192) in order to provide LPMOs, pectinases, esterases, xylanases, mannanases, feruloyl esterases and other activities. Supplementation studies based on the screening of several fungal secretomes are useful tools to identify organisms whose secretomes could improve the degradation of plant-based substrates when added to a reference T. reesei cocktail (see Table 3). In several cases, the study of the activity and composition of the best performing secretomes has led to hypotheses on the identity of the enzymes responsible for an improved performance, thus becoming ideal targets for further studies.

Other strategies enabling to identify specific enzymes rather than working with whole secretomes could be explored in the future (see Figure 2). The use of statistical analysis and algorithms could be of interest to find correlations between hydrolysis performances and complex enzymatic compositions (193,194). Another strategy to identify key enzymes in a given process is the secretome fractionation using various classical chromatography methods followed by hydrolysis tests and proteomic analyses of the best performing fractions (195,196). The identification of proteins that can supplement the secretomes of T. reesei and other industrial fungi could be an important step for the rational engineering of these microorganisms using CRISPR/Cas9 and other genome editing systems (119,197).

Other strategies that could be envisioned include the design of enzymatic cocktails by mixing in optimized proportions several secretomes obtained form various fungal species (198) or from the same species cultivated under various conditions (199), and strains co-cultivation in order to improve extracellular enzyme production and promote synergistic interactions (200-204). 404

Conclusion

In recent years, the advances in -omic technologies and the growing interest in fungal enzymes have led to multiple studies to better understand the complex arsenals of extracellular enzymes secreted by fungi under a variety of environmental conditions, from the industrial workhorses to new fungal strains isolated from biodiversity. The ability of these organisms to produce fine-tuned biomass-degrading enzyme cocktails have already led them to be used as providers of biocatalysts for industrial processes in various fields, including the textile industry, food and feed manufacturing, paper pulping, or biofuels production. However, the development of an ecologically compatible bio-based economy will require the optimization and customization of enzymatic cocktails. In this view, new sources of enzymes and new enzymatic synergies still need to be explored, discovered and exploited, and a better understanding of fungal enzymes regulation and secretion mechanisms is still needed.

Funding

Not applicable.

Figure 1 :

 1 Figure 1 : Principle of shotgun proteomics using LC-MS/MS (41), applied to fungal secretomics

Figure 2 :

 2 Figure 2 : Proposed pathways for the discovery of novel enzymes able to improve biomass degradation.

Table 1 : Secretomic studies involving T. reesei strains grown on various carbon sources.

 1

	T. reesei
		Proteomic method	Carbon sources	Enzymes reported	Source
	strains
		2-DE + MALDI-TOF MS	Lactose	22 proteins (18 CAZymes)
	CL847				(45)
		and nano LC-MS/MS	Lactose + xylose	18 proteins (15 CAZymes)
			Lactose	35 proteins (12 CAZymes)
		2-DE + MALDI-TOF MS
	RUT-C30		Xylose + arabinose +	32 proteins (9 CAZymes)	(46)
		and ESI-LC-MS/MS
			galactose + acetic acid
		2-DE + MALDI-MS and	Lactose	15 proteins (13 CAZymes)
	RUT-C30				(50)
		ESI-LC MS/MS	Sugarcane molasses	14 proteins (13 CAZymes)
			Cellulose
	QM6a and	SDS-PAGE + LC-MS/MS		More than 215 proteins
			Saw dust		(47)
	RUT-C30	with iTRAQ labeling		(including at least 90 CAZymes)
			Corn stover
			Glucose
		SDS-PAGE + LC-MS/MS
	QM6a and		Cellulose	More than 175 proteins
		with PAMUS			(48)
	RUT-C30		Starch	(including at least 85 CAZymes)
		quantification
			Starch + cellulose
			Sugarcane culm and
	RUT-C30	SDS-PAGE + LC-MS/MS	pretreated sugarcane	66 proteins (45 CAZymes)
			bagasse

  ® SmartClear cocktail from DSM. It is constituted by an A. niger secretome containing cellulases, hemicellulases, polygalacturonases, pectin methylesterase and pectin lyase enzymatic activities enriched with an endopolygalacturonase from a self-cloned A. niger strain (125). Viscozyme ® L is another enzyme cocktail produced by Novozymes from a secretome of A. aculeatus. It contains, among other enzymes, exo-and endogalacturonases and other pectinases, displaying a high transfructosylation activity when sucrose is used as substrate and is therefore of great interest for the production of FOS (126). The same cocktail was also used for pectin oligosaccharides production by hydrolysis of sugar beet pulp demonstrating that the multiple enzyme activities present in a given cocktail allow different applications (127). Other pectinase cocktails such as Rohapect Addition of the enzyme complex to the diet led to the degradation of arabinoxylans and increased digestibility of starch, fat and crude proteins by an improved accessibility of the nutrients to amylolytic, proteolytic and lipolytic enzymes. More recently, the Rovabio ® cocktail was supplemented with Aspergilli secretomes to improve soybean meal digestibility (130).

	and selection and displaying enhanced xylanase and cellulase activities. A proteomic analysis of this cocktail
	employing three complementary approaches (2D electrophoresis, ion exchange chromatography followed by
	1D-SDS-PAGE and separation of peptides after trypsin digestion by two-dimensional liquid chromatography)
	revealed the presence of at least 50 different proteins, most of the enzymatic activities corresponding to
	glycosyl hydrolases, essentially cellulases and hemicellulases (123). The efficiency on broiler feed digestibility
	of the Rovabio

®

10 L, which is produced by AB Enzymes using an A. niger strain, can also find applications in protoplast isolation from fungi or plants, and reduction of plant cell wall viscosity for food or bioenergy applications. Deeper analysis of this cocktail (128) further revealed high pectin methylesterase and polygalacturonase activities, and wide stability across varying pH.

Another important application domain for fungal secretomes rich in CAZymes is animal feed. Cereals are energy-rich, but also contain non-starch polysaccharides which restrict access to nutrients and decrease digestibility. To hydrolyze these complex compounds, fungal secretomes such as Rovabio ® , produced by Adisseo, provide multiple and complementary activities. The Rovabio ® Excel cocktail is a secretome from a Talaromyces versatilis (formerly Penicillium funiculosum) strain resulting from several rounds of mutagenesis ® Advance cocktail, produced by a genetically-modified T. versatilis strain with enhanced xylanase and arabinofuranosidase activities, was investigated by

Cozannet et al. (129)

. The arabinoxylan content of different cereal meals was negatively correlated to the digestibility and the apparent metabolizable energy of the diets.

Table 2 : Examples of commercial enzymes of fungal origin used in various biotech applications.

 2

	336
	Main application	Name	Fungal organism	Major activities	Company	Source
	Feed	Allzyme ®	Aspergillus niger Endo-1,4-xylanase	Alltech	(136)
		Rovabio ®	Talaromyces	Endo-1,4-xylanase	Adisseo	(123)
			versatilis	β-1,4-xylosidase
				Arabinofuranosidase
				Alpha-galactosidase
				Cellobiohydrolase
				Polygalacturonase
	Textile, feed	Fungal Amylase Aspergillus	Alpha-amylase	American	(137)
			oryzae			Biosystems
	Textile	Primafast ®	Trichoderma	Endoglucanase	Genencor	(136)
			reesei
	Laundry	Celluzyme ®	Trichoderma	Cellulase		Novozymes (138)
			reesei	Hemicellulase
	Beverages	Rapidase ®	Aspergillus niger Polygalacturonase	DSM	(125)
				Alpha-
				arabinofuranosidase
		Pectinex ®	Aspergillus	Polygalacturonase	Novozymes (139)
		UltraSP	aculeatus
		Rohapect ® 10L	Aspergillus niger Polygalacturonase	AB	(140)
				Pectin	Methyl	Enzymes
				esterase
				Arabinase
		Rohament ® PL		Polygalacturonase	AB	(140)
						Enzymes
	Food and	Viscozyme ® L	Aspergillus	Endo-β-glucanase	Novozymes (141)
	beverages		aculeatus	Arabanase

Table 3 : Examples of supplementation of T. reesei enzymatic cocktails with fungal secretomes

 3

	Reference cocktail	Best performing fungal	Conditions of hydrolysis Hydrolysis results	Enzymes responsible for	Source
		secretome			yield improvement
	Celluclast ®	Corynascus thermophilus	Steam-pretreated barley	Cellulose conversion yield	Endoglucanases more	(181)
	(Novozymes), 1,2 mg/g	fermentation broths (20%	straw (2% w/v), 6h,	< 55% with Celluclast + NS	active on cellulose than
	of substrate + SP188 (β-	wt of total protein load)	pH 5, 50°C	188 alone, > 70% when C.	those present in
	glucosidase from A.			thermophilus broth is added	Celluclast
	niger, Novozymes), 1
	mg/g substrate
	Spezyme ® (Genencor/	Aspergillus fumigatus	Steam-exploded corn	Glucan-to-glucose	High β-glucosidase and	(182)
	Dupont), 4.2 FPU/g of	ECU0811 grown on corn	stover (0,5% w/v), 24h,	conversion increased from	xylanase activities
	glucan	cob (1.21 FPU/g glucan)	pH 4.8, 50°C	25.6% to 99.5%
	T. reesei CL847 cocktail	Ustilago maydis grown	Micronized wheat straw	20% increase in glucose	Arabinofuranosidases	(183)
	(SAF-ISIS E508),	on autoclaved maize bran	(1% w/v), 24h, 37°C	release	and putative glucose-
	0,3 mg/g of substrate				methanol-choline
					oxidoreductases


Ramona Borsa

Michèle Freche

Jean-Louis Lacout

Georgeta Cosmeleata

Sorin Ciuca

ETUDE DE LA SYNTHESE D'UNE BIOCERAMIQUE EN APATITE SILICATEE PAR REACTION SOLIDE-SOLIDE

Silicated apatite bioceramic synthesis by solid state route. Calcium phosphate hydroxyapatite compound (HA: Ca 10 (PO 4 ) 6 (OH) 2 ) is an isostructural model of the non stoichiometric phosphocalcium apatite which is present in the mineral phases of the bone. Recent studies showed that silicon clearly influences the process of bone mineralisation. This work deals with the synthesis of silicon containing apatite by a high temperature method. Different initial mixtures with increasing amounts of silicon were investigated. Several reaction temperatures were tested between 1100°C and 1400°C with a heating rate of 5°C/min and various plateau durations. The reaction products were characterised by X-Ray diffraction and Fourier-Transform Infrared spectroscopy.The stable compound at high temperature seems to be silicocarnotite (Ca 10 (PO 4 ) 4 (SiO 4 ) 2 ). Nevertheless, a silicon-containing apatite has been obtained by heating a mixture of tri-calcium phosphate, calcium carbonate and silicon dioxide at 1200°C-1300°C. The synthesised materials can be used in medicine as bone substitutes or for the coating of prosthesis and implants.

Résumé -L'hydroxyapatite phosphocalcique HA: Ca 10 (PO 4 ) 6 (OH) 2 est un composé modèle isostructural des apatites phosphocalciques non stoechiométriques présentes dans les phases minérales osseuses. Des études récentes ont montré que le silicium influe nettement sur le processus de biominéralisation osseuse. Ce travail porte sur l'étude de la synthèse d'une apatite silicatée par réaction solide-solide à haute température. Plusieurs mélanges initiaux avec différentes quantités de silicium ont été testés. Le cycle de calcination a été réalisé à différentes températures comprises entre 1100°C et 1400°C, avec une vitesse de chauffage de 5°C/min et plusieurs durées de calcination. Les produits de réaction ont été caractérisés par diffraction des rayons X et spectroscopie infrarouge (FTIR). A haute température (supérieure à 1300°C) le composé stable est la silicocarnotite (Ca 10 (PO 4 ) 4 (SiO 4 ) 2 ). Une apatite silicatée a pu être obtenue par chauffage d'un mélange de phosphate tricalcique, de carbonate de calcium et de silice à 1200°C-1300°C. Ce matériau peut être utilisé en orthopédie comme comblement osseux, recouvrement d'implants ou de prothèses osseuses.

INTRODUCTION

Les céramiques à base de phosphates de calcium sont étudiées et utilisées depuis de nombreuses années pour leur bonne biocompatibilité et leur bioactivité [START_REF] Elliott | Structure and chemistry of the apatite's and other calcium orthophosphates[END_REF][START_REF] Driskell | Characterisation and performance of calcium phosphate coatings for implants[END_REF][START_REF] Driessens | [END_REF]. Parmi les différents phosphates de calcium, l'hydroxyapatite a fait l'objet d'une attention particulière des chercheurs et des médecins. L'hydroxyapatite phosphocalcique (HA : Ca 10 (PO 4 ) 6 (OH) 2 ) est un composé modèle isostructural des apatites phosphocalciques non stoechiométriques présentes dans les phases minérales osseuses. Elle est ainsi utilisée pour des applications médicales telles les comblements osseux, le remplissage des cavités osseuses ou les implants vertébraux et aussi pour le recouvrement des prothèses de hanche ou implants dentaires [START_REF] Heimann | [END_REF].

Dans l'os, l'hydroxyapatite présente différentes substitutions ioniques, par exemple le sodium, le manganèse, les carbonates ou les silicates [5,6]. Le comportement in vivo de l'apatite synthétique et l'influence des différents éléments de substitution ont été étudiés par plusieurs auteurs [7,8,9]. L'effet de la présence du silicium dans le processus de bio-minéralisation osseuse a été mis en évidence dès les années 1970 par Carlisle [9]. Les avantages liés à la présence de cet élément sont aussi démontrés par la bioactivité reconnue des verres à base de silicium [START_REF] Hench | Introduction in Bioceramics[END_REF][START_REF] Ohtsuki | [END_REF]. Une étude menée par Thian sur la réponse des ostéoblastes à mis en évidence l'influence du silicium sur le processus de biominéralisation [12].

Depuis 2000, plusieurs méthodes de synthèse de phosphates de calcium silicatés ont été proposées, soit par voie de précipitation en milieu aqueux, en milieu hydrothermal ou non [13][14][15][16][17] soit par voie solide-solide [8,[START_REF] Boyer | Synthèse et caractérisation d'apatites phospho-silicatées aux terres rares : application au nucléaire[END_REF]. Des études antérieures réalisées par Boyer et Lacout avaient été menées afin d'obtenir une apatite silicatée par réaction solide-solide. Dans ce cas, la compensation de charge ionique est obtenue par l'introduction des ions de terres rares, tels le La 3+ ou le Nd 3+ . Le produit final possède une grande stabilité chimique et présente de nombreuses possibilités d'application [START_REF] Boyer | Synthèse et caractérisation d'apatites phospho-silicatées aux terres rares : application au nucléaire[END_REF].

Ce travail a pour but l'étude de la synthèse de l'hydroxyapatite silicatée (HA-Si) à haute température par réaction à l'état solide, sans apport d'éléments extérieurs pour effectuer la compensation de charge. Les groupements SiO 4 4-substituent les groupements PO 4 3-dans le réseau de l'hydroxyapatite et l''équilibre de charge électrique est obtenu par la formation de lacunes d'ions OH -dans les tunnels. La formule chimique attendue est donc : Ca 10 (PO 4 ) 6-x (SiO 4 ) x (OH) 2-x . Différents mélanges initiaux qui conduisent à une même formule chimique finale sont possibles. Cependant leur réactivité et leur mise en oeuvre peuvent être différentes. Ainsi, plusieurs mélanges et plusieurs taux de silicium ont été étudiés. L'objectif a été de déterminer le mélange optimal et de décrire l'influence de différents paramètres sur le processus de synthèse.

Les applications de ces composés silicatés peuvent être diverses. Bien entendu, dans le cas où on envisage leur utilisation dans des applications biologiques telles des comblements osseux ou des revêtements de prothèses et implants osseux, le matériau céramique en apatite silicatée doit induire un phénomène de biointégration.

PROCEDURE EXPERIMENTALE

Les produits initiaux qui sont la source de calcium, phosphore et silicium peuvent être divers. Ces produits ont été choisis en tenant compte essentiellement de leur facilité d'obtention, soit par synthèse, soit directement dans le commerce, et de M2:

(6-x)/2 Ca 2 P 2 O 7 + x SiO 2 + (x+4) CaCO 3 = Ca 10 (PO 4 ) 6-x (SiO 4 ) x (OH) 2-x (2)
H 2 O -CO 2 M3: (6-x)/2 Ca 3 (PO 4 ) 2 + x Ca SiO 3 + (x+2)/2 CaCO 3 = Ca 10 (PO 4 ) 6-x (SiO 4 ) x (OH) 2-x (3)
H 2 O -CO 2 M4: (6-x)/2 Ca 2 P 2 O 7 + x Ca SiO 3 + 4 CaCO 3 = Ca 10 (PO 4 ) 6-x (SiO 4 ) x (OH) 2-x (4) H 2 O
-CO 2

Plusieurs compositions avec différents taux de silicium ont été testées, avec x compris entre 0 et 2, où x est le nombre de groupements ioniques PO 4 3-substitués par SiO 4 4-comme annoncé dans la formule générale. La compensation de charge électrique est réalisée par les lacunes dans les tunnels en remplacement des groupements OH -, donc x peut atteindre la valeur maximale de 2.

Le mélange de matériaux a été effectué dans un mortier en agate. Les cycles de calcination ont été réalisés à plusieurs températures situées entre 1100°C et 1400°C avec une vitesse de chauffage de 5°C/min et une durée de calcination comprise entre 60 minutes et 12 heures.

Après refroidissement lent, les produits ont été analysés par diffraction des rayons X en utilisant un appareil INEL avec détecteur circulaire et anticathode de cobalt. Les diagrammes obtenus ont été comparés avec les fiches standard de la base de données JCPDS (Joint Committee and Powder Diffraction Standards).

Les analyses par spectrométrie infrarouge ont été réalisées avec un appareil Nicolet FTIR-ATR avec une résolution spectrale de 4 cm -1 . Les spectres ont été obtenus entre 450 et 4000 cm -1 après 64 scans et exploités avec le logiciel OMNIC.

RESULTATS

La réaction à haute température est influencée par plusieurs paramètres :

-Les différentes compositions initiales : M1, M2, M3, M4 qui ont été précédemment décrites.

-La quantité de SiO 2 introduit, de 0 à 12 % massique.

-La température de chauffage qui varie entre 1100°C et 1400°C; -La durée de maintien en température qui varie entre 60 minutes et 12 heures; Une première étude a été réalisée pour les mélanges dans lesquels x=1, chauffés à 1200°C pendant 3 heures. Cette température a été choisie en fonction de résultats préalables rapportés dans la littérature [8,[START_REF] Boyer | Synthèse et caractérisation d'apatites phospho-silicatées aux terres rares : application au nucléaire[END_REF]. Sur la figure 1 sont présentés les diagrammes de diffraction des rayons X obtenus pour les quatre mélanges initiaux (M1, M2, M3, M4) traités. On remarque que la nature des produits initiaux influe nettement sur les résultats des traitements. Dans le cas des mélanges M1 et M2, pour lesquels l'apport de silicium est fait avec la silice, les diagrammes de diffraction des rayons X correspondent à une phase pure quasi d'apatite. Seules apparaissent quelques raies de faible intensité qui correspondent aux traces de -TCP et de CaO qui n'ont pas encore réagi. Lorsque le silicium est introduit à partir d'un silicate de calcium (M3, M4), les diagrammes de diffraction des rayons X montrent la présence de trois différents composants : hydroxyapatite, silicocarnotite (Ca 10 (PO 4 ) 4 (SiO 4 ) 2 ) et -TCP. On n'obtient une apatite quasi-pure qu'à partir des mélanges M1 et M2.

Afin d'optimiser les conditions de synthèse de cette apatite, le mélange M1 a été étudié plus précisément. Dans le tableau I sont reportées les différentes compositions pondérales des mélanges initiaux selon la valeur de x choisie, comprise entre 0 et 2. A 1200°C, après un temps de palier de 3 heures, le diagramme est celui de l'hydroxyapatite avec des traces de -TCP et CaO. A 1300°C, le résultat est identique. La seule différence observée concerne la transformation allotropique du phosphate tricalcique : -TCP en phosphate tricalcique -TCP. A plus haute température on observe la formation de la silicocarnotite.

Tableau

  C C C  α α α C S A A A A A A A A A A A A α A A
On doit remarquer que l'on n'observe dans aucun cas la formation d'oxyde de silicium, que ce soit du quartz  à température inférieure à 1300°C, ou de la cristobalite qui est la forme stable de SiO 2 à température supérieure à 1300°C. Des mesures antérieures ont montré que la présence d'oxyde de silicium, quartz  ou cristobalite, peut être mise en évidence dans un mélange avec l'hydroxyapatite lorsque sa teneur est supérieure à 3%. On peut donc supposer que la réaction attendue a eu lieu et que les silicates sont présents dans le réseau apatitique avec formation d'une silicoapatite.

Les substitutions dans l'apatite induisent des modifications du réseau cristallin. Pourtant, le calcul des paramètres de maille par raffinement Rietvelt ne révèle pas de modifications significatives. La variation des paramètres n'est dans ce cas pas facile à mettre en évidence : en effet les différentes substitutions, SiO 4 pour PO 4 dans le squelette apatitique et O = et lacune pour OH - dans les tunnels, peuvent conduire à des augmentations ou diminutions qui ne vont pas obligatoirement dans un même sens et dont les effets peuvent s'annuler.

Sur les spectres FTIR (figure 3), les bandes intenses à 1090, 1045 et 958 cm -1 correspondent aux vibrations d'élongation P-O, tandis que les bandes à 600 et 568 cm -1 correspondent aux vibrations angulaires O-P-O. Les bandes à 3570 cm -1 et 630 cm -1 sont caractéristiques des ions hydroxyles dans l'hydroxyapatite. . Effectivement, sur les spectres IR des mélanges pour x=0, x=1, x=2 on note qualitativement l'augmentation de l'intensité relative de cette bande avec la quantité de SiO 2 . Le diagramme de diffraction des rayons X n'a mis en évidence aucune phase silicatée supplémentaire, on peut donc attribuer cette bande à 883 cm -1 aux ions SiO 4 4-dans la structure de l'apatite silicatée [START_REF] Tian | [END_REF].

Pour le cas x=2, qui correspond à la formation de la silicocarnotite, un déplacement des bandes PO 4 3-et SiO 4 4-peut être observé. Ce fait est dû aux environnements différents dans la structure cristalline entre l'apatite (hexagonale) et la silicocarnotite (orthorhombique). Une bande à 875 cm -1 attribuée à SiO 4 4-est présente, mais aussi d'autres bandes à 845 cm -1 et 507 cm -1 . La présence des bandes de SiO Les observations qui ont été faites au moyen des diagrammes de diffraction des rayons X et des spectres IR permettent de conclure qu'une apatite silicatée a été obtenue à partir du mélange M1 par calcination à des températures comprises entre 1200 et 1300°C.

DISCUSSION

Si l'on observe l'ensemble des résultats, on peut remarquer que, quels que soient les mélanges initiaux, on obtient d'une manière générale :

 dans le cas x=0, une hydroxyapatite.  dans le cas x=2, un produit final qui est essentiellement constitué par de la silicocarnotite.

 dans les cas x=0,5 et x=1, un produit final qui contient soit une apatite silicatée, soit un mélange apatite-silicocarnotite.

La synthèse d'une apatite silicatée pure par réaction solide-solide est donc possible. C'est le cas pour le mélange M1 avec x=1 à 1200°C et 1300°C. On peut supposer que dans des conditions particulières de chauffage et de refroidissement et de nature des matières premières, la cinétique des réactions intermédiaires permet la formation de cette apatite silicatée. Mais bien entendu, le chauffage de cette silicoapatite à plus haute température peut conduire à une démixtion en deux phases principales : la silicoapatite et la silicocarnotite.

Le comportement en température de l'hydroxyapatite phosphocalcique a été étudié par plusieurs auteurs [20][21][22]. Les réactions qui se produisent sont bien connues. L'hydroxyapatite Ca 10 (PO 4 ) 6 (OH) 2 est stable entre 1000/1100°C. D'ailleurs, il est possible de la préparer par voie solide-solide à cette température, en présence de vapeur d'eau. Au dessus de 1100°C, même en présence de vapeur d'eau, on observe l'élimination d'eau formée par condensation des ions hydroxyle et formation de l'oxyhydroxyapatite, selon la réaction :

Ca 10 (PO 4 ) 6 (OH) 2 Ca 10 (PO 4 ) 6 O x (OH) 2(1-x) + x H 2 O (5)
A 1320°C intervient la décomposition thermique partielle de l'oxyhydroxyapatite, avec la formation de -TCP et de CaO :

Ca 10 (PO 4 ) 6 O x (OH) 2(1-x)  Ca 3 (PO 4 ) 2 + CaO (6)
Ces réactions sont réversibles et l'apatite décomposée à haute température peut être recomposée lors du refroidissement pour autant que la vitesse de refroidissement soit assez lente. Dans certain cas cette recomposition n'est pas totale, et il subsiste des phases telles que le TCP, qui peut se trouver soit sous forme , métastable, soit sous forme  stable à basse température.

Dans nos conditions expérimentales, pour x=0, l'hydroxyapatite a été obtenue après calcination des mélanges M1 et M2 entre 1300°C et 1400°C.

Dès lors que du silicium est présent dans les mélanges, la formation de la silicocarnotite Ca 10 (PO 4 ) 4 (SiO 4 ) 2 peut être observée. Cependant la formation de ce composé semble dépendre des mélanges initiaux. Ainsi, dans le cas des mélanges M1 and M2, dans lesquels le silicium est apporté sous forme de silice, la silicocarnotite n'apparait qu'après chauffage à 1400°C. Par contre, dans le cas des mélanges M3 et M4, dans lesquels le silicium est apporté sous forme CaSiO 3 , la silicocarnotite est observée dès 1200°C.

La présence de la silicocarnotite à haute température est due, dans le premier cas (voie des mélanges M1 et M2) à la décomposition de la silicoapatite formée dès 1200°C et dans le second cas (voie de mélanges M3 et M4) à la réaction avec le CaSiO 3 .

Rabu [START_REF] Rabu | Contribution à l'étude d'une hydroxyapatite frittée: Préparation et propriétés mécaniques du[END_REF]

Après traitement thermique à 1300°C, l'auteur a observé que seulement dans le cas de la réaction (8) la phase majoritaire est la silicocarnotite, donc la réaction (8) entre phosphate tricalcique et silicate bicalcique se produit plus facilement que la réaction (7).

Steinke [START_REF] Steinke | Sol-gel derived chemically bonded ceramics in the system CaO-SiO 2 -P 2 O 5 -H 2 O[END_REF] a noté par ailleurs que la formation de la silicocarnotite semble inhibée par la présence d'eau ou d'ions hydroxyle.

Ainsi on peut proposer que, dans le domaine 1100°C-1200°C, en présence d'oxyde de silicium, c'est la silicoapatite qui se forme en premier. L'obtention de cette apatite hydroxylée semble empêcher la synthèse du Ca 10 (PO 4 ) 4 (SiO 4 ) 2 au cours du traitement thermique. Au dessus de 1300°C, la phase stable formée est une silicocarnotite.

Figure 1 .

 1 Figure 1. Diagrammes de diffraction des rayons X pour les différents mélanges initiaux après chauffage à 1200°C pendant 3heures: A-hydroxyapatite, S-silicocarnotite, - TCP; - TCP; Coxyde de calcium

Figure 2 .

 2 Figure 2. Diagrammes de diffraction des rayons X des mélanges M1 pour x=1 après chauffage à différentes températures : A-hydroxyapatite,  --TCP;  --TCP; S-silicocarnotite ; C-oxyde de calcium

Figure 3 .

 3 Figure 3. Spectres FTIR des mélanges M1 après chauffage à 1300°C Il apparaît en outre une bande à 883 cm -1 . Cette bande peut être attribuée aux vibrations Si-O dans SiO 4 4-

4 4 -

 4 est associée avec l'absence des bandes OH -. Cela correspond à la limite de substitution selon le couple (des taux de silicium inférieurs à x=2, on peut proposer une substitution couplée dans la structure apatitique Cette supposition est en corrélation directe avec la formule attendue pour la silicoapatite Ca 10 (PO 4 ) 6-x (SiO 4 ) x (OH) 2-x x

  Le pyrophosphate de calcium est obtenu à partir du phosphate calcique di-hydraté (CaHPO 4 2H 2 O) chauffé à 700°C. Le Ca 3 (PO 4 ) 2 , le CaHPO 4 2H 2 O, ainsi que le silicate de calcium CaSiO 3 et SiO 2 ont été fournis par Prolabo. Le CaCO 3 utilisé est un carbonate de calcium précipité Scoralite.

	 à la stoechiométrie de la réaction. Le carbonate de calcium (CaCO 3 ) qui permet d'apporter le calcium nécessaire
		Le phosphate tricalcique a été préparé par calcination à 900°C du produit commercial afin
	d'obtenir la forme β-TCP. Les réactions chimiques attendues pour les quatre mélanges M1, M2, M3 et M4 sont :
	M1:	H 2 O
		(6-x)/2 Ca 3 (PO 4 ) 2 + x SiO 2 +(3x+2)/2 CaCO 3 -CO 2	= Ca 10 (PO 4 ) 6-x (SiO 4 ) x (OH) 2-x	(1)
		leur taux de pureté. Ce sont :
	 (Ca 2 P 2 O 7 ) pour l'apport de calcium et de phosphore ; Le phosphate tricalcique (Ca 3 (PO 4 ) 2 ) et le pyrophosphate de calcium  Le dioxyde de silicium (SiO 2 ) et le silicate de calcium (CaSiO 3 ) pour le silicium ;

  I. Pourcentage massique des matières premières avec des taux de silicium différents dans le mélange M1, avec rapport Ca/(P+Si) constant =1,667 Dans le cas du mélange sans silicium (x = 0), le diagramme de diffraction des rayons X après calcination à 1100°C, 12 heures est celui d'un mélange HA, TCP et CaO. Après traitement à une température de 1400°C pendant 1h le diagramme présente exclusivement les raies de diffraction de l'HA.Le mélange de type M1, contenant différents taux de silicium, a été chauffé à différentes températures comprises entre 1100°C et 1400°C. Dans la figure 2 sont présentés les diagrammes de diffraction des rayons X du mélange M1 avec x=1. Pour toutes les températures le composant principal formé après chauffage est l'hydroxyapatite. A 1100°C la réaction est avancée, mais n'est pas complète, même après 12 heures de maintien à la température de réaction.

	x	Ca 3 (PO 4 ) 2	SiO 2	CaCO 3
	0	90.29	-	9.71
	0,5	80.61	2.84	16.55
	1	71.43	5.53	23.04
	2	54.39	10.52	35.09

  a étudié la formation de la silicocarnotite par réaction du silicate dicalcique (Ca 2 SiO 4 ) avec soit l'hydroxyapatite phosphocalcique, soit le phosphate tricalcique selon les réactions : 2 Ca 10 (PO 4 ) 6 (OH) 2 + 3 Ca 2 SiO 4 => 3 Ca 10 (PO 4 ) 4 (SiO 4 ) 2 + Ca(OH) 2 (7) et Ca 3 (PO 4 ) 2 + Ca 2 SiO 4 => Ca 10 (PO 4 ) 4 (SiO 4 ) 2

CONCLUSION

Cette étude a montré qu'une apatite silicatée Ca 10 (PO 4 ) 6-x (SiO 4 ) x (OH) 2-x peut être synthétisée notamment par calcination de mélanges TCP-SiO 2 -CaCO 3 à des températures comprises entre 1200°C et 1300°C. Pour des températures plus élevées on observe l'apparition de la silicocarnotite.

L'obtention de la silicocarnotite semble être favorisée par la présence du silicate de calcium dans les mélanges initiaux.

Ces résultats montrent que si l'on veut obtenir une apatite silicatée, il est nécessaire de contrôler précisément la composition des mélanges initiaux et la température. Ceci est particulièrement important lors de la fabrication de matériaux destinés à l'orthopédie soit sous forme d'implants frittés, soit sous forme de revêtement d'implants métalliques.