The art and design of functional metagenomic screens

Use of Substrate-Induced Gene Expression in Metagenomic Analysis of an Aromatic Hydrocarbon-Contaminated Soil

“…Overall, the sequences of the clones characterized in this study reveal that SIGEX is capable of recovering genes that are functionally related to the chemical used for their induction. While the literature has focused on the high-throughput nature of FCM and its ability to rapidly analyze millions of clones (14,15), another substantial advantage, i.e., single-cell analysis of gene expression (34,49), has often been overlooked. Single-cell analysis improves the detection of differences between populations having unusual distributions of gene expression.…”

“…SIGEX is a promoter trap method based on single-cell sorting of clones from a plasmid library using flow cytometry (FCM), where metagenomic clones of interest are identified by the increased expression of a downstream fluorescent reporter gene in the presence, but not in the absence, of an inducing compound. SIGEX was initially perceived as having great potential for mining genes from metagenomic samples in a high-throughput manner, without requiring prior knowledge of the sequences being screened for (13)(14)(15). However, SIGEX, and metagenomic promoter traps in general, has not lived up to this potential.…”

Contemporary molecular tools in microbial ecology and their application to advancing biotechnology

“…GHs and lipase/esterases constitute the highest proportion among those enzymes Daniel 2009, Steele, Jaeger et al 2009) possibly due to the rapid and easy plate-based detection assays for these two types of enzymes (Taupp, Mewis et al 2011). The lack of availability of assay methods or more specifically of high-throughput screening methods for enzymes other than hydrolases/lipases has impeded the discovery of more biotechnologically important enzymes.…”

“…Crude-cell-lysate-based methods usually have low throughput and are less reproducible (Felczykowska, Dydecka et al 2014). Lipases/esterases constitute a major fraction of enzymes derived from metagenomic screening simply due to the availability of easy high-throughput screening methods (Taupp, Mewis et al 2011, Reyes-Duarte, Ferrer et al 2012. Apart from these direct screening methods, more sophisticated highthroughput screening technologies have recently been developed.…”

“…Knowledge of the environmental parameters underlying samples may aid in the selection of suitable screen targets and substrates for metagenomic functional screening (Taupp, Mewis et al 2011). For example, clone libraries from the bovine rumen metagenome are a logical source for carbohydrate active enzymes (CAZymes) (Patel, Patel et al 2014).…”