Métagénomique fonctionnelle du microbiote du rumen bovin pour la découverte d’enzymes de dégradation de polymères naturels et synthétiques

Bovine rumen microbiota is a highly diverse and efficient ecosystem for the degradation of complex substrates, especially those issued from plant biomass. Predominantly composed of uncultivated microorganisms, it constitutes a rich reservoir of new enzymes of potential interest for industrial biotechnologies, especially biorefineries and bioremediation. As part of this thesis, we used the functional screening of the ruminal metagenome to increase the discovery of enzymes able to degrade lignocelluloses, as well as different synthetic pollutants. In particular, new esterases able to degrade a carbamate insecticide, fenoucarb, and a commercial polyurethane, Impranil DLN, have been identified. Moreover, the development of a new screening strategy for oxidoreductases allowed the isolation of three original bacterial enzymes that are very polyspecific, and do not need copper nor manganese to degrade different polycyclic substrates, like major pollutants of the textile industry, as well as lignin derivatives. Finally, the screening of two libraries from in vivo and in vitro enrichments of the ruminal microbiome on wheat straw allowed the isolation of lignocellulolytic enzymatic cocktails, with different functional profiles and taxonomical origins, comprising glycoside-hydrolases, esterases and oxidoreductases. Fifteen novel CAZy modules, related to enzymatic families never characterized, were identified. All these results highlight the vast potential of microbial ecosystems, in particular the bovine rumen microbiota, for biotechnological innovationLe microbiote du rumen bovin est un écosystème très diversifié et efficace pour la dégradation de substrats complexes, notamment issus de la biomasse végétale. Composé majoritairement de microorganismes non cultivés, il constitue un réservoir très riche de nouvelles enzymes d’intérêt potentiel pour les biotechnologies industrielles, en particulier les bioraffineries et la bioremédiation. Dans le cadre de cette thèse, nous avons mis en œuvre une approche de criblage fonctionnel du métagenome ruminal pour accélérer la découverte d’enzymes de dégradation des lignocelluloses, mais aussi de divers polluants synthétiques. En particulier, de nouvelles estérases capables de dégrader un insecticide de la famille des carbamates, le fenobucarb, ainsi qu’un polyuréthane commercial, l’Impranil DLN, ont pu être identifiées. De plus, le développement d’une nouvelle stratégie de criblage d’oxydoréductases nous a permis d’isoler trois enzymes bactériennes originales, très polyspécifiques, ne requérant ni cuivre ni manganèse pour dégrader différents substrats polycycliques tels que des polluants majeurs de l’industrie textile, mais aussi des dérivés de lignine. Enfin, le criblage de deux banques issues d’enrichissements in vivo et in vitro du microbiome du rumen sur paille de blé a permis d’isoler des cocktails d’enzymes lignocellulolytiques au profil fonctionnel et d’origine taxonomique différents, constitués de glycoside-hydrolases, estérases et oxydoréductases. Quinze nouveaux modules CAZy, correspondant à des familles enzymatiques jamais caractérisées, ont été identifiés. L’ensemble de ces résultats met en lumière l’immense potentiel d’innovation biotechnologique contenu dans les écosystèmes microbiens, en particulier dans le microbiote du rumen bovi

Découverte de nouvelles fonctions et familles protéiques : nouveaux défis pour les biotechnologies et l’écologie microbienne

absen

Discovery of new protein families and functions: new challenges in functional metagenomics for biotechnologies and microbial ecology

The rapid expansion of new sequencing technologies has enabled large-scale functional exploration of numerous microbial ecosystems, by establishing catalogs of functional genes and by comparing their prevalence in various microbiota. However, sequence similarity does not necessarily reflect functional conservation, since just a few modifications in a gene sequence can have a strong impact on the activity and the specificity of the corresponding enzyme or the recognition for a sensor. Similarly, some microorganisms harbor certain identified functions yet do not have the expected related genes in their genome. Finally, there are simply too many protein families whose function is not yet known, even though they are highly abundant in certain ecosystems. In this context, the discovery of new protein functions, using either sequence-based or activity-based approaches, is of crucial importance for the discovery of new enzymes and for improving the quality of annotation in public databases. This paper lists and explores the latest advances in this field, along with the challenges to be addressed, particularly where microfluidic technologies are concerned

Discovery of new protein families and functions: new challenges in functional metagenomics for biotechnologies and microbial ecology

The rapid expansion of new sequencing technologies has enabled large-scale functional exploration of numerous microbial ecosystems, by establishing catalogs of functional genes and by comparing their prevalence in various microbiota. However, sequence similarity does not necessarily reflect functional conservation, since just a few modifications in a gene sequence can have a strong impact on the activity and the specificity of the corresponding enzyme or the recognition for a sensor. Similarly, some microorganisms harbor certain identified functions yet do not have the expected related genes in their genome. Finally, there are simply too many protein families whose function is not yet known, even though they are highly abundant in certain ecosystems. In this context, the discovery of new protein functions, using either sequence-based or activity-based approaches, is of crucial importance for the discovery of new enzymes and for improving the quality of annotation in public databases. This paper lists and explores the latest advances in this field, along with the challenges to be addressed, particularly where microfluidic technologies are concerned

Metagenomics for the discovery of pollutant degrading enzymes

Organic pollutants, including xenobiotics, are often persistent and toxic organic compounds resulting from human activities and released in large amounts into terrestrial, fluvial and marine environments. However, some microbial species which are naturally exposed to these compounds in their own habitat are capable of degrading a large range of pollutants, especially poly-aromatic, halogenated and polyester molecules. These microbes constitute a huge reservoir of enzymes for the diagnosis of pollution and for bioremediation. Most are found in highly complex ecosystems like soils, activated sludge, compost or polluted water, and more than 99% have never been cultured. Meta-omic approaches are thus well suited to retrieve biocatalysts from these environmental samples. In this review, we report the latest advances in functional metagenomics aimed at the discovery of enzymes capable of acting on different kinds of polluting molecules. (C) 2015 Published by Elsevier Inc

Functional metagenomics: construction and high-throughput screening of fosmid libraries for discovery of novel carbohydrate-active enzymes

Activity-based metagenomics is one of the most efficient approaches to boost the discovery of novel biocatalysts from the huge reservoir of uncultivated bacteria. In this chapter, we describe a highly generic procedure of metagenomic library construction and high-throughput screening for carbohydrate-active enzymes. Applicable to any bacterial ecosystem, it enables the swift identification of functional enzymes that are highly efficient, alone or acting in synergy, to break down polysaccharides and oligosaccharides

Le Courrier

06 mars 18511851/03/06 (N65)

Highly promiscuous oxidases discovered in the bovine rumen microbiome

The bovine rumen hosts a diverse microbiota, which is highly specialized in the degradation of lignocellulose. Ruminal bacteria, in particular, are well equipped to deconstruct plant cell wall polysaccharides. Nevertheless, their potential role in the breakdown of the lignin network has never been investigated. In this study, we used functional metagenomics to identify bacterial redox enzymes acting on polyaromatic compounds. A new methodology was developed to explore the potential of uncultured microbes to degrade lignin derivatives, namely kraft lignin and lignosulfonate. From a fosmid library covering 0.7 Gb of metagenomic DNA, three hit clones were identified, producing enzymes able to oxidize a wide variety of polyaromatic compounds without the need for the addition of copper, manganese, or mediators. These promiscuous redox enzymes could thus be of potential interest both in plant biomass refining and dye remediation. The enzymes were derived from uncultured Clostridia, and belong to complex gene clusters involving proteins of different functional types, including hemicellulases, which likely work in synergy to produce substrate degradation