Appl Microbiol Biotechnol (2012) 93:1005–1020 medium. Since only successful recombinant clones would be able to catabolize D-lysine, unsuccessful clones would starve on the medium. Using this method, a positive clone was identified and sequenced. To confirm that the inserted gene was derived from the metagenome and not from digested DNA fragments during library construction, primers based on the detected lyr (lysine racemase) gene were designed. These primers then successfully amplified the lyr gene direct- ly from the metagenomic DNA by PCR (Chen et al. 2009). Induction by substrate Induction of gene expression by substrate is particularly practical for the detection of catabolic genes. Expression of catabolic genes is often induced by substrates and/or metabolites of catalytic enzymes. The regulatory elements of these catabolic genes are generally situated close to the genes themselves. These elements have been shown to work in host organisms like E. coli. Substrate-induced gene expression Uchiyama et al. (2005) developed the so-called substrate-induced gene expression (SIGEX) system for use as a screening method for particular catabolic genes. To make this method high-throughput- compatible, an operon-trap GFP expression vector was used, resulting in co-expressed GFP upon substrate-induced expres- sion of any responsive inserted gene. This GFP expression subsequently enabled the separation of positive clones by fluorescence-assisted cell sorting (FACS). In the study, metagenomic genes from groundwater were found that could be induced by benzoate and naphthalene. This yielded 58 benzoate- and four naphthalene-positive clones (Uchiyama et al. 2005). By changing the substrate, the scope of the screen can be adjusted and different catabolic genes can be targeted. Possibly unknown gene functions might even be deduced from the inducing substrate that is used. However, induction of expression by other effectors than the substrate used can result in the detection of false positives. Metabolite-regulated expression Metabolite-regulated ex- pression constitutes a similar technique to the above one (Williamson et al. 2005). It aims to detect biologically active small molecules by an intracellular luxI–luxR biosensor system. In this system, gene expression will be induced or inhibited by quorum sensing. After a certain concen- tration of gene product is met, induction of expression of a transcriptional activator will take place by binding of luxR, which activates luxI; subsequent expression of a reporter gene will ensue. The successful expression of the reporter gene allows high-throughput screening by FACS. An advantage of this system is that it does not rely on secretion of a gene product like screens with indicator organisms do (a common technique to identify 1013 antibiotics). Instead this technique screens intracellularly for expressed small molecules (Williamson et al. 2005). Major hurdles and future prospects in functional metagenomics Functional screening will remain an essential element to be developed further in metagenomics aimed at mining natural microbial communities for new products for biotechnology. Two main hurdles can still be identified, i.e., the successful expression of genes in metagenomic clone libraries and the subsequent screening and selection of genes of interest from expressed inserts. The metagenomic expression paradigm Two facets are important in the expression of foreign genes in a metagenomics library host, i.e., the nature of the DNA insert and that of the expression machinery (consisting of a vector and host with tunable genetic circuitry). In “traditional” foreign gene expression by genetic modification using known source and host genetic backgrounds, the two facets have become relatively well known. Thus, we have learned that the host expression machinery needs to be chosen and tuned to the requirements of the specific genetic insert. Often, the insert was first codon-optimized for the expression host to maximize the chance of successful gene expression. In contrast, in functional metagenomics a new expression paradigm is encountered, in which very little is known of the genetic insert prior to gene expression. Hence, prior knowledge on the estimated prevalence of source genes in the metagenomic library can be extremely helpful and may be required. On the basis of such knowledge, suitable host/vector systems can be selected. And, in addition to that, the host expression machinery might be tuned to a broad range of inserts, with varying fragment types, origins and sizes. It seems to be the relatively narrow-range expression machinery present in E. coli which is preventing an effective match on the individual insert level, leading to the current expression bottlenecks that are often encountered in functional metagenomic screenings. Broader expression systems might overcome this bottleneck. There is still a need for the development of additional expression hosts from less studied phyla and of robust shuttle and induction systems for these hosts. But even with a broadly applicable expression system, trans-acting processes together with the shear ran- domness of insertions into the vectors used will continue to limit our ability to optimize the rate of expression of the target genes present in the metagenome. In Fig. 2, an overview of the different expression stages that are met in functional metagenomic screens is provided.