Functional metagenomics reveals novel β-galactosidases not predictable from gene sequences

Abstract

The techniques of metagenomics have allowed researchers to access the genomic potential of uncultivated microbes, but there remain significant barriers to determination of gene function based on DNA sequence alone. Functional metagenomics, in which DNA is cloned and expressed in surrogate hosts, can overcome these barriers, and make important contributions to the discovery of novel enzymes. In this study, a soil metagenomic library carried in an IncP cosmid was used for functional complementation for β-galactosidase activity in both Sinorhizobium meliloti (α-Proteobacteria) and Escherichia coli (γ-Proteobacteria) backgrounds. One β-galactosidase, encoded by six overlapping clones that were selected in both hosts, was identified as a member of glycoside hydrolase family 2. We could not identify ORFs obviously encoding possible β-galactosidases in 19 other sequenced clones that were only able to complement S. meliloti. Based on low sequence identity to other known glycoside hydrolases, yet not β-galactosidases, three of these ORFs were examined further. Biochemical analysis confirmed that all three encoded β-galactosidase activity. Lac36W_ORF11 and Lac161_ORF7 had conserved domains, but lacked similarities to known glycoside hydrolases. Lac161_ORF10 had neither conserved domains nor similarity to known glycoside hydrolases. Bioinformatic and structural modeling implied that Lac161_ORF10 protein represented a novel enzyme family with a five-bladed propeller glycoside hydrolase domain. By discovering founding members of three novel β-galactosidase families, we have reinforced the value of functional metagenomics for isolating novel genes that could not have been predicted from DNA sequence analysis alone.

Fig 1. Functional metagenomics selection/screen and characterization of novel β-galactosidases.

Fig 2. Conserved domains (E < 0.01) in β-galactosidases isolated from 12AC metagenomic library clones.

Fig 3. Biochemical characterization of novel β-galactosidases.

pH profiles of Lac161_ORF10 (A), Lac161_ORF7 (C), Lac36W_ORF11 (E). Temperature profiles of Lac161_ORF10 (B), Lac161_ORF7 (D), Lac36W_ORF11 (F).

Fig 4. Bioinformatic characterization of a putative glycosyl hydrolase domain in Lac161_ORF10.

(A) The NCBI Conserved Domain Database (CDD) predicts Lac161_ORF10 as a divergent member of the GH_J clan of glycosyl hydrolases. (B) Structural model of Lac161_ORF10 generated by Phyre 2.0, with a predicted cluster of 8 ligand-binding residues highlighted in yellow. The putative binding site was predicted by 3dLigandSite based on the Phyre model with PDB ID 1vkd (chain A) as the template. A NAG ligand is shown in red, which approximates the location of a lactose molecule in Lac161_ORF10. (C) An alignment of Lac161_ORF10 with the most similar members of the CDD’s GH_J sequence cluster (Genbank accession numbers are included on the right of the tree). The most conserved columns are coloured light blue. A predicted active site feature (D197) is highlighted in yellow, and is consistent with 3dLigandSite’s predicted cluster of ligand-binding residues.

Fig 5. Protein homology searches of novel β-galactosidase sequences of Lac161_ORF10, Lac161_ORF7 and Lac36W_ORF11 against aquatic, human gut, and soil metagenomic databases, normalized to the rpoB gene.