Appl Microbiol Biotechnol (2012) 93:1005–1020 The alternative to the upscaling of metagenomic screenings is to restrict the scope of the study by narrowing down the insert gene source diversity so that the expression machinery can be more specifically engineered to suit the experimental demands. To confine the scope of a metagenomics screen, ecological enhancement can be very useful as it decreases library complexity by an increase of the prevalence of genes of preselected and dominating target microbes. One could also think of preselecting genetic material on the basis of G–C content by ultracentrifugation (Holben 2011), thereby providing a better match between the sampled metagenome and the expression host. Heterologous complementation is also a very suitable detection method in such focused screens because of its specificity and independence of successful insert expression. A more radical approach would be to tune the selected target genes in the metagenome library to match the most convenient expression machinery of the host. This would require sequencing of a metagenomic library to identify target genes and subsequent optimization of the codons and signal sequences of these to suit the expression host. Finally, optimized sequences would be synthesized, inserted, and functionally screened in the relevant host. Computational, sequence-based, and high-throughput technologies may allow us to codon-optimize and synthesize a complete metagenomic operon for functional screening. The possibilities are enormous, like accessing ever rarer genes, as 1015 well as increasing positive hit rates. However enticing this idea may be, as well as its potential possibilities, currently only selections of existing libraries are realistic candidates for such experiments. New technologies useful in functional screenings The recent growth of the use of “library based” approaches in metagenomics-based mining is directly related to the rapid technological developments in molecular biotechnology. Screening methodologies have evolved in the light of the necessity to understand complex ecological and biochemical interactions in different environments. The original screenings, based on the isolation of mutant (transformed) cells of host organisms on culture media, were considered to provide insufficient power or throughput (Link et al. 2007; Shuman 2003). Thus, screening systems able to rapidly identify the presence and activity of enzymes, effects of mutations, or interaction and changes among microbial community members were desired. Microarray (chip)-based technologies coupled with microfluidic devices, cell compartmentalization, flow cytometry, and cell sorting have been proposed as promising new technologies (Link et al. 2007; Ottesen et al. 2006; Tracy et al. 2010). It is important to pinpoint the major roles fluorescence-based assays are playing in single-cell analyses. Improved enzyme detection, resulting from the discovery of new genes, isolation of proteins with Table 1 Examples of enzymes and other molecules derived from (meta)genomics-related methodologies Product Cellulase Lipase Protease Amylase Chitinase Fluorescent protein Antibiotics Xylanase Applicationa Textile industry, plant biotechnology Cleaning industry, academic Alkaline tolerant Food industry Pharmaceuticals, food industry, bioremediation, biomedicine Biometabolites, pharmaceutical industry for drug discovery Medicine Paper and textile industry Manufacturerb Syngenta Mogen B.V. Gist-Brocades N.V. Roche Vitamins Inc Genecor Sinobis BASF Sukahan Biotechnology Diversa Libragen, Kosan Technologies Huzhou Llilly biology Technology Co. Ltd Number of patentsc 4,735d 6,649e 10,000f 5,208e 876d 10,000g 10,000h 1,321d a The most important applications are listed here. For all products, the academic research is included as an application b Other manufacturers may be involved in the production of similar biomolecules c According to the FreePatentsOnline web engine (http://www.freepatentsonline.com/), the number of available patents related to query advance search (including US Patents, US Patent Applications, EP Documents, Abstract of Japan, and WIPO from all years) scores from 1,000 to 10 d Scores of matches from 1,000 to 10 e Scores of matches from 999 to 10 f Scores of matches from 1,000 to 74 g Scores of matches from 1,000 to 213 h Scores of matches from 1,000 to 60