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Functional metagenomics is an essential and effective approach to recover new enzymes from the environment. In this chapter, we describe a procedure to construct metagenomic library to discover new N-acyl homoserine lactone (AHL) degrading enzymes based on a direct method or an indirect enrichment procedure. Applicable to any bacterial ecosystem, it enables rapid identification of functional enzymes effective to degrade AHLs., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The vast majority of the Earth's biological diversity are hidden in uncultured and yet uncharacterized microbial genomes. The construction of metagenomic libraries is one cultivation-independent molecular approach to assess this unexplored genetic reservoir. High numbers of novel biocatalysts have been identified by function-based or sequence-based screening of metagenomic libraries derived from various environments. Here, we describe detailed protocols for the construction of metagenomic small-insert and large-insert libraries in plasmids and fosmids, respectively, from environmental DNA., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Antimicrobial resistance (AMR) is an increasingly important global challenge for healthcare systems as well as agricultural food production systems. Our ability to prepare for, and respond to, emerging AMR threats is dependent on our knowledge of genes able to confer AMR that are circulating within various environmental, animal, and human microbiomes. Targeted, sequence-specific, detection of AMR genes and functional resistance assays, described here, carried out on metagenomic DNA gives us unique insights into the presence of AMR genes and how these are associated with mobile genetic elements that may be responsible for their dissemination and can also provide important information about the mechanisms of resistance underpinning the phenotype., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Metagenomics offers the possibility to study the microbial community of soils at large scale, allowing the characterization also of unculturable microorganisms. Availability of high-quality DNA is a prerequisite of this approach. However, since soils have highly heterogeneous physicochemical properties, several parameters need to be considered for the development of the best molecular practices. A method for isolation of high-molecular-weight and good-quality metagenomic DNA from different soil samples is described here. The protocol combines physical and chemical strategies to ensure efficient cell lysis and precipitation of humic impurities-free DNA suitable for downstream processing for metagenomics study., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The vast majority of the Earth's biological diversity is hidden in uncultured and yet uncharacterized microbial genomes. The construction of metagenomic libraries is a cultivation-independent molecular approach to assess this unexplored genetic reservoir. High numbers of novel biocatalysts have been identified by function-based or sequence-based screening of metagenomic libraries derived from various environments. Here, we describe detailed protocols for the construction of metagenomic small-insert and large-insert libraries in plasmids and fosmids, respectively, from environmental DNA.

Archaea constitute one of the three recognized phylogenetic groups of organisms living on the planet, and the latest to be discovered. Most Archaea resist cultivation and are studied using molecular methods. High-throughput amplicon sequencing and metagenomic approaches have been key in uncovering hitherto unknown archaeal diversity, their metabolic potential, and have even provided an insight into genomes of a number of uncultivated members of this group. Here, we summarize protocols describing sampling, molecular, metagenomic, and metatranscriptomic analyses as well as bioinformatics approaches that have proved useful for the study of archaea in natural samples.

Activity-based metagenomics is one of the most efficient approaches to boost the discovery of novel biocatalysts from the huge reservoir of uncultivated bacteria. In this chapter, we describe a highly generic procedure of metagenomic library construction and high-throughput screening for carbohydrate-active enzymes. Applicable to any bacterial ecosystem, it enables the swift identification of functional enzymes that are highly efficient, alone or acting in synergy, to break down polysaccharides and oligosaccharides.