A phylogenetic and metagenomic study of elephant feces samples (derived from a three-weeks-old and a six-years-old Asian elephant) was conducted in order to describe the microbiota inhabiting this large land-living animal. The microbial diversity was examined via 16S rRNA gene analysis. We generated more than 44,000 GS-FLX+454 reads for each animal. For the baby elephant, 380 operational taxonomic units (OTUs) were identified at 97% sequence identity level; in the six-years-old animal, close to 3,000 OTUs were identified, suggesting high microbial diversity in the older animal. In both animals most OTUs belonged to Bacteroidetes and Firmicutes. Additionally, for the baby elephant a high number of Proteobacteria was detected. A metagenomic sequencing approach using Illumina technology resulted in the generation of 1.1 Gbp assembled DNA in contigs with a maximum size of 0.6 Mbp. A KEGG pathway analysis suggested high metabolic diversity regarding the use of polymers and aromatic and non-aromatic compounds. In line with the high phylogenetic diversity, a surprising and not previously described biodiversity of glycoside hydrolase (GH) genes was found. Enzymes of 84 GH families were detected. Polysaccharide utilization loci (PULs), which are found in Bacteroidetes, were highly abundant in the dataset; some of these comprised cellulase genes. Furthermore the highest coverage for GH5 and GH9 family enzymes was detected for Bacteroidetes, suggesting that bacteria of this phylum are mainly responsible for the degradation of cellulose in the Asian elephant. Altogether, this study delivers insight into the biomass conversion by one of the largest plant-fed and land-living animals.

Cellulose is an important renewable resource for the production of bioethanol and other valuable compounds. Several ionic liquids (ILs) have been described to dissolve water-insoluble cellulose and/or wood. Therefore, ILs would provide a suitable reaction medium for the enzymatic hydrolysis of cellulose if cellulases were active and stable in the presence of high IL concentrations. For the discovery of novel bacterial enzymes with elevated stability in ILs, metagenomic libraries from three different hydrolytic communities (i.e. an enrichment culture inoculated with an extract of the shipworm Teredo navalis, a biogas plant sample and elephant faeces) were constructed and screened. Altogether, 14 cellulolytic clones were identified and subsequently assayed in the presence of six different ILs. The most promising enzymes, CelA2, CelA3 (both derived from the biogas plant) and CelA84 (derived from elephant faeces), showed high activities (up to 6.4 U/mg) in the presence of 30% (v/v) ILs. As these enzymes were moderately thermophilic and halotolerant, they retained 40% to 80% relative activity after 34 days in 4 M NaCl, and they were benchmarked with two thermostable enzymes, CelA from Thermotoga maritima and Cel5K from a metagenome library derived from Avachinsky crater in Kamchatka. These enzymes also exhibited high activity (up to 11.1 U/mg) in aqueous IL solutions (30% (v/v)). Some of the enzymes furthermore exhibited remarkable stability in 60% (v/v) IL. After 4 days, CelA3 and Cel5K retained up to 79% and 100% of their activity, respectively. Altogether, the obtained data suggest that IL tolerance appears to correlate with thermophilicity and halotolerance.

The functional detection of novel enzymes other than hydrolases from metagenomes is limited since only a very few reliable screening procedures are available that allow the rapid screening of large clone libraries. For the discovery of flavonoid-modifying enzymes in genome and metagenome clone libraries, we have developed a new screening system based on high-performance thin-layer chromatography (HPTLC). This metagenome extract thin-layer chromatography analysis (META) allows the rapid detection of glycosyltransferase (GT) and also other flavonoid-modifying activities. The developed screening method is highly sensitive, and an amount of 4 ng of modified flavonoid molecules can be detected. This novel technology was validated against a control library of 1,920 fosmid clones generated from a single Bacillus cereus isolate and then used to analyze more than 38,000 clones derived from two different metagenomic preparations. Thereby we identified two novel UDP glycosyltransferase (UGT) genes. The metagenome-derived gtfC gene encoded a 52-kDa protein, and the deduced amino acid sequence was weakly similar to sequences of putative UGTs from Fibrisoma and Dyadobacter. GtfC mediated the transfer of different hexose moieties and exhibited high activities on flavones, flavonols, flavanones, and stilbenes and also accepted isoflavones and chalcones. From the control library we identified a novel macroside glycosyltransferase (MGT) with a calculated molecular mass of 46 kDa. The deduced amino acid sequence was highly similar to sequences of MGTs from Bacillus thuringiensis. Recombinant MgtB transferred the sugar residue from UDP-glucose effectively to flavones, flavonols, isoflavones, and flavanones. Moreover, MgtB exhibited high activity on larger flavonoid molecules such as tiliroside. For more than a decade, metagenome research has demonstrated that it is a powerful tool for the discovery of novel biocatalysts and other valuable biomolecules by using either function-or sequence-based screening technologies (1-3). Sequencebased approaches allow the identification of candidate genes. In particular, the development of next-generation sequencing (NGS) technology and improved bioinformatic tools have significantly advanced this methodology (4). However, a major drawback of sequence-based screening technologies is that they do not allow direct conclusions about the functionality and biochemical parameters of the encoded enzymes. Furthermore, sequence-based searches are limited to the identification of homologs of already known motifs (5). Yet another problem associated with the sequence-based approach is that it often reveals only partial genes, which make subsequent expression and detailed biochemical analysis of the gene products difficult if not impossible. In contrast, the function-driven approach is usually much slower and more labor-intensive and costly but results in the detection of complete and active enzyme clones. It is of course well known that function-driven metagenomics is hampered due to the problems of expressi...

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