Dorrazehi, G. M.; Penner, M.; Athanasiou, C.; Boursinhac, L.; Mobarec, J. C.; Webster, C.; Papworth, M.; Hollfelder, F.

As an alternative to historical enzyme isolation, metagenomic databases (e.g. MGnify) provide information on vast unculturable microbial diversity, especially from extreme environments, and constitute an enormous source of functional proteins. Conservative mining of these data by close sequence homology alone tends to identify merely different versions of known enzymes. Here we present a discovery strategy of meganucleases based on wider capture of less homologous enzymes with new function in metagenomic databases, incorporating metadata with homology, relying on cell-free expression to bypass host incompatibility and the need for purification, along with using deep sequencing for experimental assessment of substrate specificity and cleavage pattern, circumventing classical gel-based profiling. Specifically, we discovered the temperature-stable (>55 degrees C), intron-encoded LAGLIDADG meganuclease I-MG11 that recognizes a 17 base pair sequence to generate unique 4 base pair palindromic 3-prime-overhangs as the first monomeric meganuclease to produce such overhangs. Co-folding models of I-MG11 bound to DNA provide a structural context for enzyme-DNA interactions, highlighting differences from other monomeric LAGLIDADG meganucleases (e.g. I-SceI) shaped by InDels (insertion-deletions) in the DNA binding region that may cause specificity changes. Our strategy streamlines bona fide identification and annotation of meganucleases, while the unique properties of I-MG11 expand the molecular biology toolbox.