Analysing Megasynthetase Mutants at High Throughput Using Droplet Microfluidics.

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.

Biosynthetic incorporation of fluorinated amino acids into the nonribosomal peptide gramicidin S.

Fluorine is a key element in medicinal chemistry, as it can significantly enhance the pharmacological properties of drugs. In this study, we aimed to biosynthetically produce fluorinated analogues of the antimicrobial cyclic decapeptide gramicidin S (GS). However, our results show that the A-domain of the NRPS module GrsA rejects 4-fluorinated analogues of its native substrate Phe due to an interrupted T-shaped aromatic interaction in the binding pocket. We demonstrate that GrsA mutant W239S improves the incorporation of 4-fluorinated Phe into GS both in vitro and in vivo. Our findings provide new insights into the behavior of NRPSs towards fluorinated amino acids and strategies for the engineered biosynthesis of fluorinated peptides.

Proof-Reading Thioesterase Boosts Activity of Engineered Nonribosomal Peptide Synthetase.

Nonribosomal peptide synthetases (NRPSs) are a vast source of valuable natural products, and re-engineering them is an attractive path toward structurally diversified active compounds. NRPS engineering often requires heterologous expression, which is hindered by the enormous size of NRPS proteins. Protein splitting and docking domain insertion have been proposed as a strategy to overcome this limitation. Here, we have applied the splitting strategy to the gramicidin S NRPS: Despite better production of the split proteins, gramicidin S production almost ceased. However, the addition of type II thioesterase GrsT boosted production. GrsT is an enzyme encoded in the gramicidin S biosynthetic gene cluster that we have produced and characterized for this purpose. We attribute the activity enhancement to the removal of a stalled intermediate from the split NRPS that is formed due to misinitiation. These results highlight type II thioesterases as useful tools for NRPS engineering.