Tracing active members in microbial communities by BONCAT and click chemistry-based enrichment of newly synthesized proteins.

A comprehensive understanding of microbial community dynamics is fundamental to the advancement of environmental microbiology, human health, and biotechnology. Metaproteomics, defined as the analysis of all proteins present within a microbial community, provides insights into these complex systems. Microbial adaptation and activity depend to an important extent on newly synthesized proteins (nP), however, the distinction between nP and bulk proteins is challenging. The application of BONCAT with click chemistry has demonstrated efficacy in the enrichment of nP in pure cultures for proteomics. However, the transfer of this technique to microbial communities and metaproteomics has proven challenging and thus it has not not been used on microbial communities before. To address this, a new workflow with efficient and specific nP enrichment was developed using a laboratory-scale mixture of labelled Escherichia coli and unlabeled yeast. This workflow was then successfully applied to an anaerobic microbial community with initially low bioorthogonal non-canonical amino acid tagging efficiency. A substrate shift from glucose to ethanol selectively enriched nP with minimal background. The identification of bifunctional alcohol dehydrogenase and a syntrophic interaction between an ethanol-utilizing bacterium and two methanogens (hydrogenotrophic and acetoclastic) demonstrates the potential of metaproteomics targeting nP to trace microbial activity in complex microbial communities.