Identification of novel enzymes with different hydrolytic activities by metagenome expression cloning

Metagenome cloning has become a powerful tool to exploit the biocatalytic potential of microbial communities for the discovery of novel biocatalysts. In a novel variant of direct expression cloning, metagenomic DNA was isolated from compost by a modified direct lysis method, purified by size exclusion chromatography and cloned into an expression vector allowing bidirectional transcription. Transformation of Escherichia coli DH5alpha resulted in a metagenomic expression library with an average insert size of 3.2 kb. To estimate the functional diversity of the constructed library, it was screened by different approaches based on functional heterologous expression. A large number of active clones were identified, including lipolytic enzymes, amylases, phosphatases and dioxygenases. Molecular analysis of one important class of industrial biocatalysts, the lipolytic enzymes, confirmed the novelty and dissimilarity of all recovered genes, which exhibited only limited similarity to known enzymes. Equally, the novelty of another three genes encoding phosphatase or dioxygenase activity, respectively, was shown. These results demonstrate the suitability of this direct cloning approach, which comprised a dual-orientation expression vector and a simple one-step DNA purification method, for the efficient discovery of numerous active novel clones. By this means it provides an efficient way for the rapid generation of large libraries of hitherto unknown enzyme candidates which could be screened for different specific target reactions.