Functional expression of genes from metagenomic libraries is limited by various factors including inefficient transcription and/or translation of target genes as well as improper folding and assembly of the corresponding proteins caused by the lack of appropriate chaperones and cofactors. It is now well accepted that the use of different expression hosts of distinct phylogeny and physiology can dramatically increase the rate of success. In the following chapter, we therefore describe tools and protocols allowing for the comparative heterologous expression of genes in five bacterial expression hosts, namely Escherichia coli, Pseudomonas putida, Bacillus subtilis, Burkholderia glumae, and Rhodobacter capsulatus. Different broad-host-range shuttle vectors are described that allow activity-based screening of metagenomic DNA in these bacteria. Furthermore, we describe the newly developed transfer-and-expression system TREX which comprises genetic elements essential to allow for expression of large clusters of functionally coupled genes in different microbial species.
Keywords: Activity-based screening; Bacillus subtilis; Burkholderia glumae; Environmental DNA; Escherichia coli; Functional expression; Metagenomic library; Multi-host screening; Pseudomonas putida; Rhodobacter capsulatus; Shuttle vector; Transposon.