Environmental activity-based protein profiling for function-driven enzyme discovery from natural communities

Microbial communities are significant drivers of global biogeochemical cycles, yet accurate function prediction of their proteome and discerning their activityin situfor bioprospecting remains challenging. Here, we present environmental activity-based protein profiling (eABPP) as a novel proteomics-based approach bridging the gap between environmental genomics, correct function annotation andin situenzyme activity. As a showcase, we report the successful identification of active thermostable serine hydrolases by combining genome-resolved metagenomics and mass spectrometry-based eABPP of natural microbial communities from two independent hot springs in Kamchatka, Russia. eABPP does not only advance current methodological approaches by providing evidence for enzyme and microbial activityin situbut also represents an alternative approach to sequence homology-guided biocatalyst discovery from environmental ecosystems.