Analysing Megasynthetase Mutants at High Throughput Using Droplet Microfluidics**

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.

Directed Evolution of Piperazic Acid Incorporation by a Nonribosomal Peptide Synthetase**

“…When no exogenous Piz is added to the production culture, neither Piz GS nor native GS are produced, highlighting again the high selectivity for Piz over Pro in the evolved GrsB1. Cell extracts containing Piz GS were able to lyse liposomes containing a self‐quenching fluorescence dye following a previously established protocol for measuring membrane activity of GS [53] . Extracts containing PizGS permeabilized liposomes faster than extracts containing the same concentration of native GS (Figure S15) indicating that the Piz‐substitution enhances membrane activity.…”

“…After successfully switching the specificity of GrsB1, we used the engineered NRPS module to produce Piz GS in E. coli . For this purpose, the grsB1 gene in grsB was replaced with the engineered grsB1‐MWG in plasmid pSU18‐grsTAB carrying the entire GS biosynthetic gene cluster [53] . The gene cluster was then expressed heterologously in E. coli following a previously established protocol [53] while supplementing Piz to the media.…”

“…For this purpose, the grsB1 gene in grsB was replaced with the engineered grsB1‐MWG in plasmid pSU18‐grsTAB carrying the entire GS biosynthetic gene cluster [53] . The gene cluster was then expressed heterologously in E. coli following a previously established protocol [53] while supplementing Piz to the media. Surprisingly, the evolutionary intermediate GrsB1‐SQSF‐VM showed the highest production yield for Piz GS under in vivo conditions, not the mutant GrsB1‐MWG.…”