Analysing Megasynthetase Mutants at High Throughput Using Droplet Microfluidics.

Pourmasoumi F, Hengoju S, Beck K, Stephan P, Klopfleisch L, Hoernke M, Rosenbaum MA, Kries H2023Analysing Megasynthetase Mutants at High Throughput Using Droplet Microfluidics. Chembiochem 24, e202300680-e202300680.

Abstract

Nonribosomal peptide synthetases (NRPSs) are giant enzymatic assembly lines that deliver many pharmaceutically valuable natural products, including antibiotics. As the search for new antibiotics motivates attempts to redesign nonribosomal metabolic pathways, more robust and rapid sorting and screening platforms are needed. Here, we establish a microfluidic platform that reliably detects production of the model nonribosomal peptide gramicidin S. The detection is based on calcein-filled sensor liposomes yielding increased fluorescence upon permeabilization. From a library of NRPS mutants, the sorting platform enriches the gramicidin S producer 14.5-fold, decreases internal stop codons 250-fold, and generates enrichment factors correlating with enzyme activity. Screening for NRPS activity with a reliable non-binary sensor will enable more sophisticated structure-activity studies and new engineering applications in the future.

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