1016 high affinity, as well as phylogenetic analyses were reported (Aharoni et al. 2006; Feng et al. 2007; Melkko et al. 2007). Large metagenome libraries have been screened via single- cell fluorescent assays and high-throughput flow cytometry in the quest for novel catalytic activities (Link et al. 2006; Santoro et al. 2002; Varadarajan et al. 2005). These screening methods offer higher levels of quantification and the possibility to detect multiple traits in one assay. Considering the evolution of screening approaches, from Sydney Brenner’s affirmation “just toothpicks and logic” to higher- level technologies (which have become available at low cost), increases in the rates of discovery of new bio-engineered molecules can be predicted. The hunt for novelty The increasing human impact on the environment in the last century has urged developments in our food, waste treatment, agriculture, and biomedical industries. In this respect, explora- tions of diverse habitats have increased with the increasing demand for new enzymes, antibiotics, and other active biomo- lecules as well as biofuels (Ferrer et al. 2009; Lorenz and Eck 2005; Schloss and Handelsman 2003). A recently developed database—denominated MetaBioMe (Sharma et al. 2010)— offers access to 510 “commercially useful” enzymes (CUEs) by linking protein databases with data from metagenomic and bacterial genomic datasets. These CUEs have been classified into nine broad application categories, namely: agriculture, biosensor, biotechnology, energy, environment, food and nu- trition, medical, other industries, and miscellaneous. Among these, biotechnology, food and nutrition, medicine, and bio- degradation of toxic compounds are considered to be of utmost importance. So far, one of the most frequently targeted habitats for finding genetic “novelty” was soil. The cryptic microbial treasures of different types of soil and sediments, including those in extreme conditions (e.g., low pH, high temperature, high salt concentration), have promised the pres- ence of an enormous reservoir of different enzymes. Of late, such environments are certainly underexplored. However, metagenomics-derived products have already found their way to the biotechnology market (Table 1), although their metagenomic origin is not always revealed by the manufac- turer. Moreover, it is often protected by patents. Conclusion High-throughput technology has been often associated with increasing the success of function-based metagenomic screens. However, high-throughput is more of a way of compensating for the often low hit rates in metagenomics screens than a true improvement of methods. The upscaling of a functional meta- genomics screen by adopting a high-throughput strategy using Appl Microbiol Biotechnol (2012) 93:1005–1020 existing screening techniques may indeed increase the chance of identifying target genes in a metagenome, and indeed there is the rightful expectation that these high-throughput screens will become more effective by the use of micro- fluidic strategies, substrates with higher sensitivity, smartly designed induction systems, and easily detectable reporter genes. Collectively, such improvements may result in the low- ering of detection thresholds and saving of costs. Depending on the purpose of the application, there is still ample room for improvement of expression strategies, such as careful host (range) selection, co-expression of chaperones, or codon optimization. It is the latter strategy that holds great potential for the future by screening of sequence databases to identify genes of interest, after which the targeted genes are Fig. 3 The metagenomic expression bottleneck