Screening 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 the number of 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**

“…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.…”

“…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.…”

Directed evolution of piperazic acid incorporation by a nonribosomal peptide synthetase

“…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. [49] The gene cluster was then expressed heterologously in E. coli following a previously established protocol [49] while supplementing Piz to the media. Surprisingly, the evolutionary intermediate SQSF-VM showed the highest production yield for Piz GS under in vivo conditions, not the mutant GrsB1-MWG.…”

Gerichtete Evolution von Piperazinsäureeinbau durch eine Nichtribosomale Peptidsynthetase**

“…Zu diesem Zweck wurde das grsB1-Gen in grsB durch das modifizierte grsB1-MWG-Gen im Plasmid pSU18-grsTAB ersetzt, welches das gesamte GS-Biosynthesegencluster trägt. [53] Das Gencluster wurde anschließend heterolog in E. coli unter Verwendung eines zuvor etablierten Protokolls exprimiert, [53] während Piz dem Medium zugesetzt wurde. Überraschenderweise zeigte der evolutionäre Zwischenschritt GrsB1-SQSF-VM unter in vivo-Bedingungen die höchste Ausbeute an Piz , gemessen: m/z = 586.3713 [M + 2H] 2 + , Abbildung 3c).…”