Metagenomic insights into the fibrolytic microbiome in yak rumen

Abstract

The rumen hosts one of the most efficient microbial systems for degrading plant cell walls, yet the predominant cellulolytic proteins and fibrolytic mechanism(s) remain elusive. Here we investigated the cellulolytic microbiome of the yak rumen by using a combination of metagenome-based and bacterial artificial chromosome (BAC)-based functional screening approaches. Totally 223 fibrolytic BAC clones were pyrosequenced and 10,070 ORFs were identified. Among them 150 were annotated as the glycoside hydrolase (GH) genes for fibrolytic proteins, and the majority (69%) of them were clustered or linked with genes encoding related functions. Among the 35 fibrolytic contigs of >10 Kb in length, 25 were derived from Bacteroidetes and four from Firmicutes. Coverage analysis indicated that the fibrolytic genes on most Bacteroidetes-contigs were abundantly represented in the metagenomic sequences, and they were frequently linked with genes encoding SusC/SusD-type outer-membrane proteins. GH5, GH9, and GH10 cellulase/hemicellulase genes were predominant, but no GH48 exocellulase gene was found. Most (85%) of the cellulase and hemicellulase proteins possessed a signal peptide; only a few carried carbohydrate-binding modules, and no cellulosomal domains were detected. These findings suggest that the SucC/SucD-involving mechanism, instead of one based on cellulosomes or the free-enzyme system, serves a major role in lignocellulose degradation in yak rumen. Genes encoding an endoglucanase of a novel GH5 subfamily occurred frequently in the metagenome, and the recombinant proteins encoded by the genes displayed moderate Avicelase in addition to endoglucanase activities, suggesting their important contribution to lignocellulose degradation in the exocellulase-scarce rumen.

Figure 1. Organization of genes on representative contigs retrieved from the fibrolytic BAC clones.

Red, fibrolytic enzymes; Blue, regulatory proteins; Light green, membrane protein/transporter system; Grey, diverse functions; Dark green, hypothetical proteins with the domains deposited in Pfam database; Black, hypothetical protein without known domains. SP, signal peptide. (A) Contig 310-00038: a Bacteroidetes-derived sequence from a BAC clone with carboxylmethyl cellulase activity. (B) Contig 212-00025: a Bacteroidetes-derived sequence from a BAC clone with xylanase activity. (C) Contig 210-00018: a Firmicutes-derived sequence from a BAC clone with lipase activity. Lower case letters refer to the gene fragments discussed in the text. Scale indicates the nucleotide base pairs.

Figure 2. Sequence coverage of the 35 BAC-assembled contigs over the average coverage of total genes.

In the parentheses and on the top of each bar indicate the contig length and the fibrolytic related genes, respectively. Red, Bacteriodetes; Blue, Firmicutes; Brown, Fibrobacter; Grey, Unclassified.

Figure 3. Phylogenetic tree of the putative GH5 proteins encoded by genes from the BAC library.

The tree was constructed from 300 amino acid sequences using Mage 3.0 software. ACX75523 (GH45), an endoglucanase from Fibrobacter succinogenes, was used as the outgroup. Names of the organisms from which sequences are derived are given. Sequences with an “Uc” prefix refer to uncultured clones, and those with “contig” are from this study. Proteins with sequences in boldface were overexpressed and characterized in this study. Cluster affiliation of glycoside hydrolase families are given on the right, and the GenBank accession numbers follow the sequence names. Numbers at the cluster nodes are the supporting percentages of bootstrap evaluation. Bar, 20% sequence divergence.