Abstract
The natural bacterial diversity is regarded as a treasure trove for natural products. However, accessing complex cell mixtures derived from environmental samples in standardized high-throughput screenings is challenging. Here, we present a droplet-based microfluidic platform for ultrahigh-throughput screenings able to directly harness the diversity of entire microbial communities. This platform combines extensive cultivation protocols in aqueous droplets starting from single cells or spores with modular detection methods for produced antimicrobial compounds. After long-term incubation for bacterial cell propagation and metabolite production, we implemented a setup for mass spectrometric analysis relying on direct electrospray ionization and injection of single droplets. Even in the presence of dense biomass we show robust detection of streptomycin on the single droplet level. Furthermore, we developed an ultrahigh-throughput screening based on a functional whole-cell assay by picoinjecting reporter cells into droplets. Depending on the survival of reporter cells, droplets were selected for the isolation of producing bacteria, which we demonstrated for a microbial soil community. The established ultrahigh-throughput screening for producers of antibiotics in miniaturized bioreactors in which diverse cell mixtures can be screened on the single cell level is a promising approach to find novel antimicrobial scaffolds.
Highlights
The natural bacterial diversity is regarded as a treasure trove for natural products
We present a comprehensive and versatile platform allowing the investigation of cell proliferation and secondary metabolism of complex microorganisms within pL-droplets
Besides supplying appropriate conditions for cell propagation, we enabled cells to reach the physiological state for secondary metabolite production, which we inferred from the detection of secondary metabolites in pooled droplet supernatants
Summary
The natural bacterial diversity is regarded as a treasure trove for natural products. Molecule libraries coupled to beads[10] can meet these demands, since the beads concentration and their frequency at the encapsulation site can be controlled by simple dilution, leading to adjustable numbers of beads per droplet These libraries can be composed of all kinds of living cells expressing different products like antibodies[11,12,13], enzymes[14,15,16,17] or antibiotics[18]. Cells demand specific conditions during metabolite production, the overwhelming majority of screens in droplets is conducted with cell libraries of only one cell type to keep the timing synchronized and the experimental conditions simple In those cases, the libraries are constituted of diversified DNA sequences generated by mutagenesis[16,19] or metagenomic DNA fragments[18,20,21,22] introduced into undemanding expression hosts like Escherichia coli. The inability of common expression hosts to provide precursors or conduct necessary posttranslational modifications further minimizes the chance to detect such products in functional screenings[26,27]
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