Opening structural biology to enzymes from non-isolated microbes by a shotgun approach

The vast majority of microbes cannot be isolated, hindering the biochemical characterization of their enzymes, harboring native (metallo)cofactors, post-translationalmodifications, and physiological partners that can be overlooked when employing recombinant systems. These enzymes represent a treasure trove of catalysts that could be potentially applied for biomimicry to industrial applications. Here, we propose an alternative approach to bypass the microbial isolation barrier and the bias of recombinant expression by directly characterizing enzymes from complex microbial communities. In other words, we will anaerobically purify enzymes involved in transforming one-unit carbon (C1) from microbial enrichment, bioreactors, or even sludge. As a proof of concept, we isolated, crystallized, and structurally characterized the methane-generating enzyme from an activated wastewater treatment plant sludge. Future results will sharpen our view on C1-metabolism and their enzymatic mechanistics.