Activity-Based Metagenomic Screening and Biochemical Characterization of Bovine Ruminal Protozoan Glycoside Hydrolases

Abstract

The rumen, the foregut of herbivorous ruminant animals such as cattle, functions as a bioreactor to process complex plant material. Among the numerous and diverse microbes involved in ruminal digestion are the ruminal protozoans, which are single-celled, ciliated eukaryotic organisms. An activity-based screen was executed to identify genes encoding fibrolytic enzymes present in the metatranscriptome of a bovine ruminal protozoan-enriched cDNA expression library. Of the four novel genes identified, two were characterized in biochemical assays. Our results provide evidence for the effective use of functional metagenomics to retrieve novel enzymes from microbial populations that cannot be maintained in axenic cultures.To process fibrous plant materials, the rumen harbors a collection of diverse microorganisms, including bacteria, archaea, fungi, and protozoa (reviewed in references 33 and 45). While the diversity and functions of the thousands of microbial species of this unique ecosystem (32) are interesting from both the evolutionary and functional perspectives, the rumen also represents a rich resource of enzymes for converting lignocellulosic feedstocks into biofuel (35, 43) and other applications (19). A range of inexpensive, robust enzymes with a broad range of specificities will likely be required for efficient industrial processing of highly complex plant polysaccharides. Identification of such enzymes that microorganisms use to break down plant materials has been greatly facilitated by metagenomics (42), both in the form of activity-based screens (20, 52) and also through increasingly powerful, high-throughput genomic DNA sequencing approaches (28, 57). As evident in numerous studies (28,39,41), metagenomics has proven to be particularly effective for identification of carbohydrate-active genes of fiber-adherent bacterial species of the rumen.In addition to bacteria and archaea, the rumen also hosts eukaryotic species, namely, anaerobic fungi and ciliate protozoans (reviewed in reference 33). Addressing the function of ruminal protozoans, in particular, has been a challenge due to the difficulty of maintaining these organisms in axenic cultures (55). Thus, assessing the diversity and dynamics of ruminal protozoans has been addressed historically in morphogenic studies (reviewed in reference 12) and by molecular phylogenetics (e.g., by using 18S rDNA markers [47]). Ruminal protozoans are known to contribute to fiber degradation in their hosts (21), and determination and characterization of their ability to directly process plant material have been addressed by diverse strategies, such as direct, biochemical detection of specific fibrolytic enzymes (e.g., cellulases) in extracts derived from individual protozoan species (38, 54), by molecular cloning studies to directly identify genes encoding enzymes capable of degrading cellulose or hemicellulose (49, 50) and, most recently, by sequencing of protozoan-derived expressed sequence tag (EST) libraries (41). Early studies to establish the capacity of protozoan ...

Metatranscriptomic Analyses of Plant Cell Wall Polysaccharide Degradation by Microorganisms in the Cow Rumen

“…Rumen anaerobic fungi and protozoa are capable of efficient hydrolysis of cellulose, as demonstrated by biochemical assays of their cellulase activities (60)(61)(62). We show that enzymes of the GH5, GH9, and GH48 families are the predominant cel-GHs from rumen anaerobic fungi and protozoa, as reported previously (22).…”

Rumen protozoa of different ages of beef cattle raised in tropical pastures during the dry season

“…Additionally, a lower carboxymethyl cellulose activity in the rumen of defaunated lambs was observed, showing lower cellulose digestibility than those faunated (Santra and Karim 2002). Additionally, studies of metagenomic screening of glucosidases and metatranscriptomes have shown that a diverse range of glycoside hydrolases are present in the rumen ciliates (Findley et al 2011;Qi et al 2011).…”

Repeated inoculation of cattle rumen with bison rumen contents alters the rumen microbiome and improves nitrogen digestibility in cattle

“…Limited information exists on the role of rumen protozoa in fiber degradation 24, 25 , but functional protozoal glycosyl hydrolases have been identified, successfully expressed and biochemically characterized 26 . Rumen fiber degradation depends on complex interactions between bacteria, protozoa and fungi and the plant cell wall.…”