Cell-free expression and NGS-powered substrate profiling reveal a thermostable metagenomic meganuclease from an extreme environment with novel specificity

As an alternative to historical enzyme isolation, metagenomic databases (e.g. MGnify) provide information on vast unculturable microbial diversity, including 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 for meganucleases based on wider capture of less homologous enzymes in metagenomic databases, and incorporating metadata with their homology. We use cell-free expression to bypass host incompatibility and the need for purification, along with deep sequencing for experimental assessment of substrate specificity and cleavage pattern, thereby circumventing classical gel-based profiling. Specifically, we discovered the temperature-stable (>55°C), intron-encoded LAGLIDADG meganuclease I-MG11 that recognizes a 17 base pair sequence to generate 4 nucleotide palindromic 3′-overhangs — 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 general bona fide identification and annotation of meganucleases, while the unique properties of I-MG11 expand the toolbox of molecular genome engineering.