1010 and did not take into account key factors that play defined roles in successful gene expression, such as the presence of co-factors, protein folding, and/or secretion. One way to more successfully express genes in metage- nomic library hosts may be to engineer the host expression machinery on the basis of the expected prevalence of genes from source hosts. Thus, it would be interesting to tinker with the host’s transcription and translation systems, thereby increasing the recognition of the foreign RBS predicted to be prevalent in the metagenome (Bernstein et al. 2007). Moreover, one could boost the co-expression of chaperone proteins to promote proper protein folding, whereas enhance- ment of secretion of the target gene product is another possibility (Ferrer et al. 2004; Jhamb et al. 2008). Not only the host but also the vector can be engineered to maximize the rate or frequency of gene expression. An example is the use of dual-orientation promoters on the vector, which may effectively increase the rate of successful gene expression (Lämmle et al. 2007). However, such promoters are probably most useful in small-fragment libraries where native promoters may not be present in the insert. Single-host systems Most single-host metagenomic expres- sion systems rely on E. coli. This organism is readily used regarding gene expression assays based on many different vectors. Because of its status as the most well-known model host, there is ample knowledge about different useful gene expression strategies. In fact, a wide variety of expression systems is available for use in E. coli and many genetic constructs have been assayed this way. There is also a broad range of strains capable of efficient replication of such vectors, which are either single- or multi-copy, and confer low-frequency recombination and protection against lytic phages (Sørensen and Mortensen 2005). Multiple-host systems An alternative to the single-host strategy, which increases the rate of gene expression, is the use of multiple hosts, either sequentially or in parallel. The use of multiple hosts diversifies the available expression machinery, thus increasing the chance of successful gene expression. At the same time, the effect of gene product toxicity and enzymatic breakdown can be overcome. To express genes from metagenomes in multiple hosts, shuttle vectors with broad host range are of use. An example of an advanced vector design (combining key characteristics) for broad-host-range screenings was recently presented by Aakvik et al. (2009). Fosmid libraries constructed using broad-host-range fosmid and BAC vector pRS44 were successfully transferred into Pseudomonas fluorescens and Xanthomonas campestris. The main features of this vector are (1) inducible copy number for controlled gene Appl Microbiol Biotechnol (2012) 93:1005–1020 expression, which minimizes possible gene product toxicity but allows high-level gene expression for effective detection in screenings, (2) the ability to stably hold inserts of up to 200 kb, and (3) a high capacity to be efficiently transferred to a wide range of hosts (Aakvik et al. 2009). A nice example of a metagenomic study in which broad-host-range vectors were used was provided by Craig et al. (2010). Metagenomic libraries derived from soil were constructed in an IncP1-α broad-host-range cosmid vector using six selected proteobacterial host strains, i.e., Agrobacterium tumefaciens, Burkholderia graminis, Caulobacter vibrioides, E. coli, Pseudomonas putida, and Ralstonia metallidurans. Library screenings were conducted on the basis of three types of phenotypic traits: antibiosis, pigmentation, and colony morphology. Remarkably, a high diversity of expression profiles between the differ- ent hosts was found, with little overlap (Craig et al. 2010). This illustrates the fact that the same metage- nomic library can yield totally different expression data, purely based on the expression host used. Furthermore, the still rather low frequencies of clones with desired genes indicated the need for more robust screening meth- ods to lower detection thresholds. Another broad-host- range study (Martinez et al. 2004), which targeted novel drugs, had already underlined the need for multiple-host gene expression. Parallel screenings of metagenomic librar- ies in multiple hosts yielded diverse expression profiles of antibiotic- producing genes between hosts (Martinez et al. 2004). On the basis of the foregoing, we conclude that an invest- ment in the development of more sophisticated host–vector systems on the basis of a broad range of host organisms is needed. In particular, the development of host–vector systems with environmentally prevalent strains from phyla that are relatively incompatible with the E. coli expression machinery (like Acidobacteria and Verrucomicrobia) holds great poten- tial to increase the rates of expression of genes from metagenomes. Future developments Metagenomic approaches are in- creasingly being assisted by massive (directly obtained) sequence information. To bypass the difficulties of gene expression, it should be possible, on the basis of such information, to “translate” a whole coding sequence to the expression signal and optimal codon usage typical for E. coli. As an example, Bayer et al. (2009) codon- optimized 89 genes with possible relation to methyl halide transferases for expression in E. coli and obtained an impressive result. That is, 94% of the predicted genes were expressed and showed methyl halide transferase activity. This example clearly indicates the high potential of codon optimization strategies, in this case, evidenced in E. coli.