AI Article Synopsis
- Glycosylated bacteriocins, also known as glycocins, show promise as natural food preservatives and alternatives to antibiotics, but researchers need effective methods for their laboratory evolution.
- A new high-throughput screening method called SELECT-GLYCOCIN was developed, which utilizes an enzyme from Enterococcus faecalis to create and test libraries of modified glycocins against food-borne pathogens.
- Through this method, a diverse range of mutant glycocins was generated and screened, leading to the identification of several potential antimicrobial variants that could be useful in food preservation and drug development.
Article Abstract
Glycosylated bacteriocins (glycocins) are potential clean label food preservatives and new alternatives to antibiotics. Further development requires the availability of a method for laboratory evolution of glycocins, wherein the challenges to overcome include ensuring glycosylation in a heterologous host, avoiding potential toxicity of active glycocins to the host, and provisioning of a one-pot screening assay for active mutants. Employing EntS, a sequential O/S- di-glycosyltransferase from Enterococcus faecalis TX0104, a proof of the concept microbial system and high throughput screening assay (SELECT-GLYCOCIN) is developed for generation of O/S- linked glycopeptide libraries and screening of glycocins for desired activity/property. The method enabled enzyme-dependent in vivo glycosylation in the heterologous host and rapid screening of mutants of enterocin 96 (Ent96)- a glycocin active against food-borne pathogen L. monocytogenes. Using SELECT-GLYCOCIN, a library of random (1.5 X 10^3) and rational (17) mutants of Ent96 was generated. The mutants were screened for bioactivity to identify a total of 376 random and 14 rational mutants as bioactive. Downstream detailed analysis of 16 random and 14 rational mutants led to the identification of sequence- and or glyco-variants namely, G16E-H24Q, C13T, and Ent96-K4_K5insYYGNGV (PedioEnt96) as improved antimicrobials. To summaries, SELECT-GLYCOCIN provides a system and a generic method for discovery and screening of glycocins that can further be adapted to any known/unknown glycocins and can be employed in food preservatives' and drug discovery programs.
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SELECT-GLYCOCIN: a recombinant microbial system for expression and high-throughput screening of glycocins.
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Article Synopsis
- Glycosylated bacteriocins, also known as glycocins, show promise as natural food preservatives and alternatives to antibiotics, but researchers need effective methods for their laboratory evolution.
- A new high-throughput screening method called SELECT-GLYCOCIN was developed, which utilizes an enzyme from Enterococcus faecalis to create and test libraries of modified glycocins against food-borne pathogens.
- Through this method, a diverse range of mutant glycocins was generated and screened, leading to the identification of several potential antimicrobial variants that could be useful in food preservation and drug development.
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