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.
Highlights
The rumen is a unique natural habitat that has evolved into a complex and efficient system for lignocellulose degradation
Enzymatic and protein-structure studies indicated that freeenzyme system and cellulosomes are the two main lignocellulosedegrading mechanisms used by cultured fibrolytic bacteria [7]
We investigate fibrolytic genes and gene clusters in the rumen microbiome of yak fed on wheat stalk by constructing a large-insert bacterial artificial chromosome (BAC) library for the rumen metagenomic DNA and subsequent pyrosequencing of the inserts of clones active in a fibrolytic enzyme screening
Summary
The rumen is a unique natural habitat that has evolved into a complex and efficient system for lignocellulose degradation. Over the past few decades, considerable efforts have been made to isolate fibrolytic bacteria and identify lignocellulose-degrading enzymes from the rumen of a variety of herbivores. Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens, Butyrivibrio fibrisolvens, and Prevotella ruminicola are believed to be the predominant lignocellulose degraders in the rumen [1]. Complexity of the lignocellulosedegrading enzymes in these bacteria has been confirmed by genome sequencing [2,3,4]. Metagenomic studies demonstrated that in the rumen, plant cell wall-degrading enzymes exist in far greater diversity than previously believed [5,6]. Enzymatic and protein-structure studies indicated that freeenzyme system and cellulosomes are the two main lignocellulosedegrading mechanisms used by cultured fibrolytic bacteria [7]
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