Realizing the potential of microbes as cellular factories requires the development of orthogonal strategies for tuning pathway performance. In nature, spatial organization of biological functions is common, even in organisms as simple as bacteria. The capacity to engineer such spatial organization is thus an important goal in synthetic biology. Bacterial microcompartments (MCPs) are proteinaceous organelles that specifically aid in the proliferation of bacteria on niche carbon sources. These MCPs are delimited by a protein shell that encases an enzymatic core, where encapsulation of enzymes is hypothesized to confer several benefits to metabolic pathway performance, including increased pathway flux, sequestration of toxic intermediates, and access to a private cofactor pool. The genetically-encoded nature of both the core and shell of these organelles makes MCPs attractive protein engineering targets; however, the low throughput of existing MCP characterization techniques has limited the scope of many engineering efforts to date. Here, we describe our discovery that closure of the 1,2-propanediol utilization (Pdu) MCP from model pathogen <i>Salmonella enterica</i> serovar Typhimurium LT2 is mediated by vertex protein PduN. We find that, in the absence of PduN, extended Pdu microtube (MT) structures form, leading to an elongated cell phenotype that can be rapidly screened by both flow cytometry and high-throughput microscopy. We leverage this phenotype-assembly link to characterize how different point mutant libraries of PduN impact MCP closure, presenting the first high-throughput screening platform for MCP assembly.

High-throughput screening of bacterial microcompartment closure

ACS Spring 2022

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