Live Biosensors for Ultrahigh-Throughput Screening of Antimicrobial Activity against Gram-Negative Bacteria

Abstract

Gram-negative pathogens represent an urgent threat due to their intrinsic and acquired antibiotic resistance. Many recent drug candidates display prominent antimicrobial activity against Gram-positive bacteria being inefficient against Gram-negative pathogens. Ultrahigh-throughput, microfluidics-based screening techniques represent a new paradigm for deep profiling of antibacterial activity and antibiotic discovery. A key stage of this technology is based on single-cell cocultivation of microbiome biodiversity together with reporter fluorescent pathogen in emulsion, followed by the selection of reporter-free droplets using fluorescence-activated cell sorting. Here, a panel of reporter strains of Gram-negative bacteria Escherichia coli was developed to provide live biosensors for precise monitoring of antimicrobial activity. We optimized cell morphology, fluorescent protein, and selected the most efficient promoters for stable, homogeneous, high-level production of green fluorescent protein (GFP) in E. coli. Two alternative strategies based on highly efficient constitutive promoter pJ23119 or T7 promoter leakage enabled sensitive fluorescent detection of bacterial growth and killing. The developed live biosensors were applied for isolating potent E. coli-killing Paenibacillus polymyxa P4 strain by the ultrahigh-throughput screening of soil microbiome. The multi-omics approach revealed antibiotic colistin (polymyxin E) and its biosynthetic gene cluster, mediating antibiotic activity. Live biosensors may be efficiently implemented for antibiotic/probiotic discovery, environmental monitoring, and synthetic biology.

High-Level De Novo Biosynthesis of 2′-Fucosyllactose by Metabolically Engineered Escherichia coli

“…To further improve 2′-FL biosynthesis, the expression of manC - manB and gmd - wcaG in the chromosome was strengthened using a strong promoter. P J23119 is a commonly used strong constitutive promoter in synthetic biology, which has been widely used for the efficient expression of heterologous proteins. , Herein, the native promoters of gene clusters manC - manB and gmd - wcaG on the chromosome were replaced with the promoter P J23119 , yielding strains BWLWC and BWLWG, respectively. Synergistically strengthening the supply of GDP- l -fucose by switching the native promoter to P J23119 in both clusters was also conducted, yielding BWLWCG.…”

High-throughput bacterial co-encapsulation in microfluidic gel beads for discovery of antibiotic-producing strains

“…Similar approaches using microfluidic double emulsions 40–42,50 or W/O microfluidic droplet selection systems (FADS) 25 have been reported for the identification of microorganisms with bactericidal activity. However, to produce double emulsions, partially hydrophilic–hydrophobic microfluidic devices are needed, 28 as well as the introduction of flow resistance elements to achieve a single aqueous core emulsion.…”

“…40,41 The same methodology was used to identify Paenibacillus polymyxa, a soil strain with antimicrobial activity against E. coli. 42 In addition, gel beads coupled to FACS have been used to screen a metagenomic library for secreted antibiotics that kill the human pathogen S. aureus. 43 In these previous studies, using FACS to select particles with antagonistic microorganisms required the application of up to two fluorescent markers and a fluorescent reporter strain.…”

Creation of Recombinant Biocontrol Agents by Genetic Programming of Yeast

“…Osterman) and Bacillus megaterium B-512 (kindly provided by S.A. Dubiley). For generating the E. coli ΔlptD sfGFP reporter strain, E. coli ΔlptD cells were transfected with a plasmid that constitutively expressed the green fluorescent protein sfGFP [ 18 ].…”