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Researchers have recognized the potential of enzymes and metabolic pathways hidden among the unseen majority of Earth's microorganisms for decades now. Most of the microbes expected to colonize the seafloor and its subsurface are currently uncultured. Thus, their ability and contribution to element cycling remain enigmatic. Given that the seafloor covers ∼70% of our planet, this amounts to an uncalled potential of unrecognized metabolic properties and interconnections catalyzed by this microbial dark matter. Consequently, a tremendous black box awaits discovery of novel enzymes, catalytic abilities, and metabolic properties in one of the largest habitats on Earth. This mini review summarizes the current knowledge of cultivation-dependent and -independent techniques applied to seafloor habitats to unravel the role of the microbial dark matter. It highlights the great potential that combining microbiological and biogeochemical data from in situ experiments with molecular tools has for providing a holistic understanding of bio-geo-coupling in seafloor habitats and uses hydrothermal vent systems as a case example.

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Marine sponge-associated bacteria are known as bio-active compound produce. We have constructed metagenome libraries of the bacteria and developed a metagenomic screening approach. Activity-based screening successfully identified novel genes and novel enzymes; however, the efficiency was only in 1 out of 104 clones. Therefore, in this study, we thought that bioinformatics could help to reduce screening efforts, and combined activity-based screening with database search. Neutrophils play an important role for the immune system to recognize excreted bacterial by-products as chemotactic factors and are recruited to infection sites to kill pathogens via phagocytosis. These excreted by-products are considered critical triggers that engage the immune system to mount a defense against infection, and identifying these factors may guide developments in medicine and diagnostics. We focused on genes encoding amino acid ligase and peptide synthetase and selected from an in-house sponge metagenome database. Cell-free culture medium of each was used in a neutrophil chemiluminescence assay in luminol reaction. The clone showing maximum activity had a genomic sequence expected to produce a molecule like a phospho-N-acetylmuramyl pentapeptide by the metagenome fragment analysis.

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Lignin is an abundant cell wall component, and it has been used mainly for generating steam and electricity. Nevertheless, lignin valorization, i.e. the conversion of lignin into high value-added fuels, chemicals, or materials, is crucial for the full implementation of cost-effective lignocellulosic biorefineries. From this perspective, rapid screening methods are crucial for time- and resource-efficient development of novel microbial strains and enzymes with applications in the lignin biorefinery. The present review gives an overview of recent developments and applications of a vast arsenal of activity and sequence-based methodologies for uncovering novel microbial strains with ligninolytic potential, novel enzymes for lignin depolymerization and for unraveling the main metabolic routes during growth on lignin. Finally, perspectives on the use of each of the presented methods and their respective advantages and disadvantages are discussed.

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The (ab)use of designer drugs and steroid hormones has gained popularity due to the lower chance of getting caught, as routine drug or doping tests may miss these (novel) compounds. Current analytical approaches mostly make use of targeted, structure-based techniques, such as immunoassays or mass spectrometry (MS)-based methods. However, these approaches have limitations, including a lack of cross-reactivity and the need for prior knowledge of molecular identity. This has initiated considerable interest in the so-called "untargeted" screening strategies to detect these compounds. The use of "untargeted" MS-based screening methods (e.g. gas chromatography MS and especially high-resolution MS) has gained considerable interest to detect and identify novel compounds. However, due to their expensive and time-consuming character, very sophisticated analytical methods are not ideal as a first-line screening method and are not routinely implemented in most laboratories. Given the above, it is clear that there lies potential in novel "untargeted" screening approaches, which are less expensive, more high-throughput-amenable and more routinely applicable. Activity-based assays, capable of monitoring the biological activity of an abused substance in a biological matrix, have been proposed as an alternative. These biological assays do not require knowledge about a compound's structure and could be used as a first-line screening tool to identify potentially positive samples. In this review, we focus on activity-based reporter bioassays for the detection of steroids and drugs of abuse in biological matrices. As for drugs of abuse, only bioassays for detecting cannabinoid or opioid activity in biological matrices are available, only (synthetic) cannabinoid receptor agonists and opioids are discussed.

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Functional screens have been extensively used for searching native enzymes or mutant variants in clone libraries. Esterases and lipases are the most retrieved enzymes, because they are within the more demanded industrial enzymes and because a number of simple and generic screening methods can be applied for their screen. Here, we describe the use of a generic pH indicator assay protocol which unambiguously allows detecting in high-throughput manner esterase and lipase activity and quantifying specific activities using an ester concentration above 0.5 mM. The described method is simple and generic to allow the selection of esterases and lipases targeting desired esters.

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We describe a novel array on array strategy intended to enhance the throughput of enzymatic activity screening using microarrays. This strategy consists of spotting a first array with large droplets of enzymes with varying concentrations and subsequently spotting a second array with small droplets of fluorogenic substrate on top of the enzyme array. By varying the array on array spotting patterns of different classes of enzyme (e.g., proteases, phosphatases, kinases) and their corresponding fluorogenic substrates, we have the unprecedented ability for testing enzymes and mixed samples in a multiplexed fashion within a single microarray slide. This new approach enables rapid enzyme characterization building upon a one enzyme on one slide droplet-based screening concept previously established.

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Functional expression of genes from metagenomic libraries is limited by various factors including inefficient transcription and/or translation of target genes as well as improper folding and assembly of the corresponding proteins caused by the lack of appropriate chaperones and cofactors. It is now well accepted that the use of different expression hosts of distinct phylogeny and physiology can dramatically increase the rate of success. In the following chapter, we therefore describe tools and protocols allowing for the comparative heterologous expression of genes in five bacterial expression hosts, namely Escherichia coli, Pseudomonas putida, Bacillus subtilis, Burkholderia glumae, and Rhodobacter capsulatus. Different broad-host-range shuttle vectors are described that allow activity-based screening of metagenomic DNA in these bacteria. Furthermore, we describe the newly developed transfer-and-expression system TREX which comprises genetic elements essential to allow for expression of large clusters of functionally coupled genes in different microbial species.

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