Appl Microbiol Biotechnol (2012) 93:1005–1020 DOI 10.1007/s00253-011-3804-3 MINI-REVIEW The great screen anomaly—a new frontier in product discovery through functional metagenomics David Matthias Ekkers & Mariana Silvia Cretoiu & Anna Maria Kielak & Jan Dirk van Elsas Received: 12 October 2011 / Revised: 27 November 2011 / Accepted: 29 November 2011 / Published online: 22 December 2011 # The Author(s) 2011. This article is published with open access at Springerlink.com Abstract Functional metagenomics, the study of the collec- tive genome of a microbial community by expressing it in a foreign host, is an emerging field in biotechnology. Over the past years, the possibility of novel product discovery through metagenomics has developed rapidly. Thus, metagenomics has been heralded as a promising mining strategy of resources for the biotechnological and pharmaceutical industry. However, in spite of innovative work in the field of functional genomics in recent years, yields from function-based metagenomics studies still fall short of producing significant amounts of new products that are valuable for biotechnological processes. Thus, a new set of strategies is required with respect to fostering gene expression in comparison to the traditional work. These new strategies should address a major issue, that is, how to successfully express a set of unknown genes of unknown origin in a foreign host in high throughput. This article is an opinionating review of functional metagenomic screening of natural microbial communities, with a focus on the optimization of new product discovery. It first summarizes current major bottlenecks in functional metagenomics and then provides an overview of the general metagenomic assessment strategies, with a focus on the challenges that are met in the screening for, and selection of, target genes in metagenomic libraries. To identify possible screening limitations, strategies to achieve optimal gene expression are reviewed, examining the molecular events all the way from the transcription level through to the secretion of the target gene product. D. M. Ekkers : M. S. Cretoiu (*) : A. M. Kielak : J. D. van Elsas Department of Microbial Ecology, Centre for Ecological and Evolutionary Studies, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands e-mail: m.s.cretoiu@rug.nl Keywords Metagenomics . Functional screening . Natural product discovery . Gene expression Introduction One of the major hurdles in microbial ecology is the inability to culture most of the microbial diversity present in ecosys- tems under laboratory conditions. The observed divergence between the numbers of bacterial cells forming colonies on plates and the cell count obtained by microscopic examination is known as “the great plate count anomaly” (Staley and Konopka 1985). In fact, only a fraction of the microbial diversity present in most ecosystems (1–5%) can be accessed through standard cultivation techniques (Curtis and Sloan 2004; Nichols 2007; Staley and Konopka 1985; Torsvik and Øvreås 2002). Thus, we can only speculate about the environmental importance and economical value of the majority of organisms that have remained unexplored so far. To access and explore this hitherto unexplored microbiota, the genetic material of the collective cells from an environ- mental sample can be directly extracted. This microbial community DNA, also known as the metagenome, can be further analyzed using modern technologies such as screens of constructed expression libraries and direct high-throughput sequencing. The molecular analysis strategies used to examine microbial metagenomes have been denoted as metagenomics techniques. Metagenomics has come a long way since the term was first introduced by Handelsman et al. (1998). Recently, the enormous potential of metagenomics to promote both bioexploration and our understanding of ecosystems has become clear (Hil and Fenical 2010; Imhoff et al. 2011; Lefevre et al. 2008; Mocali and Benedetti 2010; Riesenfeld et al. 2004; Singh and Macdonald 2010; Warnecke and Hess 2009). Clearly,