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.

Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)

“…Functional metagenomics has been used to identify a variety of carbohydrate hydrolases (glycosidases) from environmental libraries ( 2 – 4 ). Several studies have reported discovery of novel glycosidases (cellulases, glucosidases, xylanases, etc.)…”

Modelamiento de Glicosil hidrolasas de la familia Ustilaginaceae empleando el servidor en línea SWISS-MODEL

“…Para fortalecer los resultados obtenidos se hará una examinación en los aminoácidos que integran las estructuras secundarias y/o dominios más relevantes donde se encontró una alta similitud estructural, siendo las diecisiete proteínas las de mayor interés. Además se efectuarán análisis bioinformáticos del sitio activo y de la secuencia del péptido señal donde a través de las herramientas bioinformáticas previamente mencionadas se pueden descubrir nuevas familias como lo fue para las β-galactosidasas (Cheng et al, 2017) y un estudio filogenético para ver la historia evolutiva de las diferentes familias a las que pertenecen las glicosil hidrolasas. Intra et al (2008) reportaron secuencias proteicas relacionadas a la familia numero veinte de las glicosil hidrolasas (GH20) de organismos eucariotas y procariotas.…”

Isolation of highly thermostable β-xylosidases from a hot spring soil microbial community using a metagenomic approach

“…One of the advantages of the former method is that it could find highly homologous sequences with distinct activities from vast sequences, especially encoding active and non-active enzymes, which may lead speculation of function of each amino acid residue on the enzyme activity, as shown in our results of active forms, AR19M-311-2, AR19M-311-11, and AR19M-311-21 and a non-active form AR19M-311-27, which could not be obtained by the latter method alone. Recently, isolation of novel β-galactosidases not predictable from gene sequences was reported, 17 in which the enzymes were identified from a cosmid library with ∼33 kbp metagenomic DNA in surrogate hosts by the activity-driven method. They emphasize the importance of developing of multi-host systems for functional screening to substantially extend the breadth of gene discovery.…”