Functional metagenomics to mine the human gut microbiome for dietary fiber catabolic enzymes

Abstract

The human gut microbiome is a complex ecosystem composed mainly of uncultured bacteria. It plays an essential role in the catabolism of dietary fibers, the part of plant material in our diet that is not metabolized in the upper digestive tract, because the human genome does not encode adequate carbohydrate active enzymes (CAZymes). We describe a multi-step functionally based approach to guide the in-depth pyrosequencing of specific regions of the human gut metagenome encoding the CAZymes involved in dietary fiber breakdown. High-throughput functional screens were first applied to a library covering 5.4 3 10 9 bp of metagenomic DNA, allowing the isolation of 310 clones showing beta-glucanase, hemicellulase, galactanase, amylase, or pectinase activities. Based on the results of refined secondary screens, sequencing efforts were reduced to 0.84 Mb of nonredundant metagenomic DNA, corresponding to 26 clones that were particularly efficient for the degradation of raw plant polysaccharides. Seventy-three CAZymes from 35 different families were discovered. This corresponds to a fivefold target-gene enrichment compared to random sequencing of the human gut metagenome. Thirty-three of these CAZy encoding genes are highly homologous to prevalent genes found in the gut microbiome of at least 20 individuals for whose metagenomic data are available. Moreover, 18 multigenic clusters encoding complementary enzyme activities for plant cell wall degradation were also identified. Gene taxonomic assignment is consistent with horizontal gene transfer events in dominant gut species and provides new insights into the human gut functional trophic chain.

Mannoside recognition and degradation by bacteria

“…These strategies, especially those of Bacteroides species, rely on dedicated PULs for complex glycan assimilation. These genomic loci, which have been the subject of great interest in recent years, are often exchanged between bacteria by horizontal gene transfer (Lozupone et al, 2008;Tasse et al, 2010), which may underlie their ubiquitous distribution in ecosystems.…”

“…The GH130_2 subfamily, which contains Bt1033 and Uhgb_MP (another mannoside phosphorylase belonging to an unknown human gut bacterium assigned to the Bacteroides genus), are encoded by PULs encoding other enzymes possessing activities required for degradation of mature and immature CNGs and HMNGs, namely the GH92 α-mannosidases and GH18 N -acetyglucosaminidases. Specifically, Uhgb_MP, identified by high-throughput functional screening of the human gut metagenome (Tasse et al, 2010), showed high affinity for β-d-manp-1,4-d-GlcpNAc, the core disaccharide of all N-glycans (Ladevèze et al, 2013) (Fig. 4B).…”

Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases

“…Functional metagenomic mining can be targeted to highly specific functional properties, while genome or sequence-based metagenome mining only accesses enzymes by class, without explicit specificity of properties. Few reported metagenomic screening studies have specifically targeted AcXEs, with most focusing on other CAZy classes or failing to classify identified CEs into families [116][117][118][119]. The limited focus on AcXEs is due, in part, to a lack of high throughput (HTP) metagenomic screening methods for direct detection of AcXE activity.…”

New insight into the gut microbiome through metagenomics

“…One example of such application is the discovery of new carbohydrate active enzyme (CAZyme) genes in the human gastrointestinal microbiota. The gut bacteria produce a vast amount of CAZymes in order to digest dietary fibers into metabolizable monosaccharides and disaccharides; 78,79 15,882 different CAZymes have been detected in a mini-microbiome composed of 177 sequenced members of the human gut microbiota, representing its major phylogenetic lineages. 78 Tasse et al 79 applied high-throughput functional screens and identified 310 clones showing beta-glucanase, hemicellulase, galactanase, amylase, or pectinase activities.…”