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The FibroChip, a functional DNA microarray to monitor cellulolysis and hemicellulolysis activities of rumen microbiota

ABSTRACT: Ruminants are the most efficient herbivorous animals to transform plant biomass into edible products, principally thanks to the rumen microbiota that produces a large array of enzymes responsible for the hydrolysis of plant cell wall polysaccharides. Several biotic and abiotic factors influence the efficiency of fiber degradation, which can ultimately impact the animal productivity and health. To provide more insight on mechanisms involved in the modulation of fibrolytic activity, a functional DNA microarray targeting genes coding for key enzymes involved in cellulose and hemicellulose degradation by rumen microbiota was designed. The DNA microarray, designated FibroChip, was validated using targets of increasing complexity. This new tool demonstrated sensitivity and specificity as well as explorative and semi-quantitative potential. The FibroChip was designed with the objective to propose a high throughput tool that enables to get a rapid picture of the capacity of rumen microorganisms to degrade cellulose and hemicellulose based on a targeted metatranscriptomics approach. We chose to focus on a few number of genes by targeting main ruminal fibrolytic microorganisms and selected CAZyme families that may have a pivotal role in cellulose and hemicellulose degradation. The microarray targets the coding sequence of catalytic domains from 8 CAZyme families involved in cellulose and hemicellulose degradation (i.e. glycoside hydrolases GH5, GH9, GH10, GH11, GH43 and GH48, and carbohydrate esterases CE1 and CE6). Taken together, these families present complementary activities needed for the complete degradation of cellulose and hemicellulose. In total, 392 nucleic sequences encoding 363 GH and 29 CE were kept for the microarray design. These sequences originated from 41 bacterial species, 4 protozoal species and 10 fungal species. The microarray is composed of 1631 25-mer probes. GoArrays strategy consisting by associating 2 25-mer probes targeting the same gene (Rimour et al., 2005) was also emplyed to determine 2618 composite probes of 54-mers. Triplicate of probes of 25 and 54-mers were synthetized in situ on an Agilent 8x15K DNA microarray. The microarray contained also 382 Agilent internal control probes including positive controls, negative controls and quality control probes. Probes were randomly placed on the array to avoid position bias.

ORGANISM(S): Bacteroides ovatus uncultured Neocallimastigales Bacteroides fragilis Fibrobacter succinogenes subsp. succinogenes S85 Ruminococcus albus Fibrobacter intestinalis Bifidobacterium animalis Selenomonas ruminantium Piromyces sp. Lactococcus lactis Bacteroides thetaiotaomicron Piromyces sp. 'equi' Xylanibacter ruminicola Polyplastron multivesiculatum Orpinomyces joyonii Roseburia hominis Neocallimastix patriciarum Orpinomyces sp. Acetivibrio clariflavus Bacteroides sp. Ruminococcus champanellensis Cellulosilyticum ruminicola Piromyces rhizinflatus Bifidobacterium adolescentis Neocallimastix frontalis Ruminococcus sp. Epidinium ecaudatum Enterococcus faecium Roseburia intestinalis Escherichia coli K-12 Eudiplodinium maggii Pseudobacteroides cellulosolvens Bacteroides xylanisolvens [Eubacterium] cellulosolvens Segatella bryantii Acetivibrio thermocellus Butyrivibrio hungatei Butyrivibrio fibrisolvens Bifidobacterium longum Agathobacter rectalis Cellulomonas flavigena Clostridium acetobutylicum Limosilactobacillus fermentum Enterobacter sp. Ruminococcus flavefaciens Cellulomonas fimi Bacteroides xylanisolvens XB1A Clostridioides difficile bovine gut metagenome Ruminiclostridium cellulolyticum Lachnospira eligens Clostridium beijerinckii Epidinium caudatum Fibrobacter succinogenes Clostridium cellulovorans Levilactobacillus brevis Piromyces communis Pseudobutyrivibrio xylanivorans

PROVIDER: GSE107550 | GEO | 2017/12/01

SECONDARY ACCESSION(S): PRJNA420557

REPOSITORIES: GEO

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Project description:The search for new enzymes and microbial strains to degrade plant biomass is one of the most important strategies for improving the conversion processes in the production of environment-friendly chemicals and biofuels. In this study, we report a new Paenibacillus isolate, O199, which showed the highest efficiency for cellulose deconstruction in a screen of environmental isolates. Here, we provide a detailed description of the complex multi-component O199 enzymatic system involved in the degradation of lignocellulose. We examined the genome and the proteome of O199 grown on complex lignocellulose (wheat straw) and on microcrystalline cellulose. The genome contained 476 genes with domains assigned to carbohydrate-active enzyme (CAZyme) families, including 100 genes coding for glycosyl hydrolases (GHs) putatively involved in cellulose and hemicellulose degradation. Moreover, 31% of these CAZymes were expressed on cellulose and 29% on wheat straw. Proteomic analyses also revealed a complex and complete set of enzymes for deconstruction of cellulose (at least 22 proteins, including 4 endocellulases, 2 exocellulases, 2 cellobiohydrolases and 2 -glucosidases) and hemicellulose (at least 28 proteins, including 5 endoxylanases, 1 -xylosidase, 2 xyloglucanases, 2 endomannanases, 2 licheninases and 1 endo--1,3(4)-glucanase). Most of these proteins were secreted extracellularly and had numerous carbohydrate-binding domains (CBMs). In addition, O199 also secreted a high number of substrate-binding proteins (SBPs), including at least 42 proteins binding carbohydrates. Interestingly, both plant lignocellulose and crystalline cellulose triggered the production of a wide array of hydrolytic proteins, including cellulases, hemicellulases and other GHs. Our data provide an in-depth analysis of the complex and complete set of enzymes and accessory non-catalytic proteins—GHs, CBMs, transporters, and SBPs—implicated in the high cellulolytic capacity shown by this bacterial strain. The large diversity of hydrolytic enzymes and the extracellular secretion of most of them supports the use of Paenibacillus O199 as a candidate for second-generation technologies using paper or lignocellulosic agricultural wastes.

Project description:Hemicellulose, the second most abundant plant biomass fraction after cellulose, is widely viewed as a potential feedstock for the production of liquid fuels and other value-added materials. Degradation of hemicellulose by filamentous fungi requires production of many different enzymes, which are induced by biopolymers or its derivatives and regulated mainly at the transcriptional level through transcription factors (TFs). Neurospora crassa has been shown to express and secrete plant cell wall associated enzymes. To better understand genes specifically associated with degradation of hemicellulose, we identified 353 genes by transcriptome analysis of N. crassa wild type strain grown on beechwood xylan. Exposure to xylan induces 9 of the 19 predicted hemicellulase genes. The xylanolytic phenotype of strains with deletions in genes identified from the secretome and transcriptome analysis of wild type showed that none were essential for growth on beechwood xylan. The transcription factor XlnR/Xyr1 in Aspergillus and Trichoderma species is considered to be the major transcriptional regulator of genes encoding both cellulases and hemicellulases. We identified a xlnR/xyr1 homolog in N. crassa, NCU06971, termed xlr-1 (xylanase regulator 1). Deletion of xlr-1 in N. crassa abolishes the growth on xylan and xylose, but growth on cellulose was indistinguishable from wild type. To determine regulatory mechanisms associated with hemicellulose degradation, we explored the transcriptional regulon of XLR-1 under xylose and xylanolytic versus cellulolytic conditions. XLR-1 regulated only some predicted hemicellulase genes in N. crassa and was required for a full induction of several cellulase genes. Hemicellulase gene expression was induced by a combination of release from carbon catabolite repression (CCR) and induction. However, in N. crassa, xlr-1 is subject to non-CRE-1 mediated CCR. This systematic analysis provides the similarities and differences of hemicellulose degradation and regulation mechanisms used by N. crassa in comparison to other filamentous fungi.

Project description:Plant biomass holds tremendous potential as a renewable feedstock in the production of biofuels and biochemicals. The effective co-utilization of the main components cellulose and hemicellulose in plant lignocellulose is critical to the economic viability of lignocellulosic biorefineries. Here, we found that the thermophilic cellulolytic fungi Myceliophthora thermophila can utilize cellulose and hemicellulose synchronously. To investigate the underlying molecular mechanisms, we firstly checked the soluble carbohydrate of the culture using plant biomass (corncob) as sole carbon source and revealed the presence of various oligosaccharides including cellodextrin and xylodextrin, both intracellularly and extracellularly in the cultures, in addition to glucose or xylose. Based on these, intracellular oligosaccharide metabolism was proposed and confirmed by identification of cellodextrin and xylodextrin metabolism pathway. Furthermore, sugar consumption assay showed that contrasting with synchronous utilization of mixed cello-/xylo-dextrin, the inhibition effect of glucose for the metabolism of xylose and cello-/xylo-dextrin exists in this fungus, suggesting Carbon Catabolite Repression (CCR) is largely avoided at the form of oligosaccharides. Transporter MtCDT-2 showing preference to xylobiose and the tolerance of cellobiose inhibition also helps to bypass metabolic inhibition. Finally, the expression of cellulase and hemicellulase genes, were found orthogonal induction by cellobiose/Avicel and xylobiose/xylan, which conferred the ability of the strain to synchronously utilize cellulose and hemicellulose. Taken together, the orthogonal oligosaccharide catabolic pathway in this fungus establishes the molecular basis for the synchronous utilization of cellulose and hemicellulose, which sheds new light on understanding the plant biomass degradation by fungi and provides alternative paradigm for development of lignocellulose biorefinery such as consolidated bioprocessing in the future.

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The FibroChip, a functional DNA microarray to monitor cellulolysis and hemicellulolysis activities of rumen microbiota

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