Sheep rumen shotgun sequencing for biomass-degrading genes discovery.

Abstract: The lignocellulose present in the plant biomass, is a promising source of energy generation. However, the breakdown of plant biomass into simple sugars for bioethanol production is still inefficient and costly due to the recalcitrant nature of plant fiber. Sheep rumen microbiome is specialized in degradation of plant material, but most members of this complex community are non-cultivable in laboratory. Therefore, the search for new lignocellulolytic enzymes in microbial communities naturally evolved in the biomass degradation, in environments such as the rumen, using the exploration of the metagenome, is a promising strategy for the exploration of genes. In this context, this study aimed to obtain plant biomass-degrading genes, selected from the sheep rumen microorganisms. The rumen samples were collected from 6 fistulated animals (Ovis aries), divided into two groups and subjected to two diets: control and sugarcane bagasse, 60 days after the beginning of the experiment. To characterize biomass-degrading genes, the metagenomic DNA was extracted from the solid contents of rumen followed by sequencing in MiSeq Personal Sequencer platform (Illumina®). We analyzed, 4,68 gigabases of metagenomic total DNA from microbes adherent to plant fiber, using on the CLC Genomic Workbench v.5.5.1 platform (CLC Bio, Denmark). The assembled contigs that allowed identification of 27 putative partial carbohydrate-active enzymes (CAE) (NCBI-nr) representing a total of 11 lignocellulases, 13 amylases and 3 other putative CAE from animals fed with control diet and 106 putative partial CAE representing a total of 52 lignocellulases, 46 amylases and 8 other putative CAE from animals fed with diet amended with sugarcane bagasse. These data sets shows the sheep rumen microbiome as an untapped source of potential new fibrolytic enzymes. Using a diet amended with sugarcane bagasse increases the abundance of CAE and provide a substantially expanded catalog of genes participating in the deconstruction of plant biomas

Exploring the sheep rumen shotgun sequencing for funcional analysis and lignocellulolitic enzyme discovery.

The rumen harbors complex microbial communities which participate in an efficient process to digest plant biomass. This ecosytem represents an untapped source of hydrolytic enzymes with potential application for second?generation biofuel production from lignocellulosic biomass. The search for new lignocellulolytic enzymes in microbial communities naturally evolved in the biomass degradation, in environments such as the rumen, using the exploration of the metagenome, is a promising strategy for the exploration of genes. In this context, this study aimed to describe the functions and explores the potential for lignocellulolitic enzyme in the sheep rumen microbiome. The rumen samples were collected from 6 fistulated animals (Ovis aries), divided into two groups and subjected to two diets: control and sugarcane bagasse, 60 days after the beginning of the experiment. Metagenomic DNA was extracted from the solid rumen contents and sequencing was performed in MiSeq Personal Sequencer platform (Illumina®). We analyzed, 4,68 GB of metagenomic DNA from microbes adherent to plant fiber using MGRAST metagenomics analysis server. The functional annotation was performed at MG?RAST for the total functional profile using the KEGG orthology level 2. The shotgun metagenomic reads of all animals samples was assigned to putative lignocellulolitic enzymes when considering nine protein databases at MG?RAST. The predictive functional profiling of the sheep rumen microbiome revealed that amino acid and carbohydrate metabolism, translation, DNA replication and repair, and membrane transport are dominant functions in the rumen microbiome. This functional pattern was similar across all animals. As expected, carbohydrate metabolism was highly represented in our data set, supporting the importance of the rumen microbiome for fiber degradation. Reads classification using nine databases resulted in 22 lignocellulases. For instance, the TrEMBL representing 76,77% out of a total 933639 protein abundance, followed by SwissProt representing 39,95 %, Seed 20,71%, PATRIC 5,92%, IMG 3,63%, KEEG 3,44%, GenBank 3,13%, RefSeq 3,09%, eggnog 1,48%. Based on Cazy search for glycosyl hydrolase (GH) families, more than 50 GH families were detected . The most abundant enzymes were ??glucosidase (GH1; GH30), Endo?1,4???xylanase (GH5; GH10; GH51) , ??N?arabinofuranosidase (GH7; GH51; GH54), ??galactosidase( GH27; GH31; GH36), Acetylesterase, Cellulase (GH5; GH9, GH7), Cellobiose phosphorylase (GH94), ??mannosidase(GH2; GH5), ??galactosidase(GH1; GH2; GH35). This results showing the sheep rumen microbiome as a promising source of new fibrolytic enzymes

Chromosome segments representing the organization of genes in islands (color coded arrows and note colors beneath the bars).

<p>Expression at 200 DAI (heatmap red scale) and <i>in vitro</i>(heat map blue scale) are compared using the normalized number of mapped Illumina paired end reads, represented by the scales under each chromosome island. Gene names are presented at the borders of each segment of the chromosome, numbers represent the coordinates of these islands in kbp and red dots represent singlets as defined by OrthoMCL</p

Syntenic view of two chromosomes of <i>S. reilianum</i> that merged as one in <i>S. scitamineum</i>.

<p>Links represents alignment length of more than 1 kbp obtained by BLASTn (e-value < 1 x 10^-5). The first outer circle represents the chromosome and scale is coordinates in base pairs. The second indicates the GC content followed by predicted coding regions of the plus and minus strands. Bars display the % of identity to orthologous in <i>S. reilianum</i>. The most inner circle represents the RNAseq coverage of each chromosome region. Red lines are RNAseq data of <i>S. scitamineum</i> growing in planta and blue lines growing <i>in vitro</i>. Circle images of all chromosomes are available in the Supporting Information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129318#pone.0129318.s003" target="_blank">S3 File</a>.</p

Representation of all chromosomes and contigs assembled of the strain SSC39 genome compared to the strain 2014001 scaffolds.

<p>Regions present in the strain 2014001 are shown in green blocks, delimited by black borders. White regions represent sequences unique to the strain SSC39 assembly. The analysis was performed using MUMmer 3 and parameters = -maxmatch -c -L -b -l 500. Red line above the chromosome 2 indicates the region containing the mating-type <i>loci</i></p

Blocks of synteny between chromosome 2 of <i>S. scitamineum</i> and chromosomes 1 and 20 of <i>S. reilianum</i> and schematic representation of the linked mating-type <i>loci</i> in <i>S. scitamineum</i>.

<p>Blue areas correspond to syntenic regions considering BLASTn e-value ≤ 1 x 10^-5. Red lines represent the expansion of the region containing the mating type genes in <i>S. scitamineum</i> located at positions 792,295 bp to 863,606 bp of the chromosome 2. The chromosome breakpoint is identified and indicated by a red dot above the sequence. Genes are indicated by gray arrows placed according to transcriptional orientation and the transposons related sequences are highlighted in red. Letters represent functional annotation of encoded proteins: A) <i>c1d1</i> putative nuclear regulator; B and C) homeodomain transcription factor <i>bE1</i> and <i>bW1</i>, respectively; D) <i>nat1</i> putative N-terminal acetyltransferase; E, F, M, N, P, Q and R) Uncharacterized protein; G, J, M and S) Related to transposase; H) <i>sla</i>—cytoskeleton assembly control protein; I) RPN5-26S proteasome regulatory protein; K) <i>hhp1</i> casein kinase-1; L) related to reverse transcriptase; O) <i>arp2/3</i>—actin related protein 2/3 complex; T) <i>lba1</i> left border <i>a</i><i>locus</i>; U) and V) pheromone gene <i>mfa1.2</i> and <i>mfa1.3</i>, respectively; W) <i>pra1</i> pheromone receptor gene; X) <i>Rba2</i>—right border <i>a locus</i>; Y) <i>pan1</i>—pantoate-beta-alanine ligase.</p