@article{open2623,
           month = {November},
           title = {SELECT-GLYCOCIN: a recombinant microbial system for expression and high-throughput screening of glycocins},
          author = {Pravinkumar Choudhary and Alka Rao},
       publisher = {Springer},
            year = {2020},
            note = {The copyright of this article belongs to Springer.},
         journal = {Glycoconjugate Journal  },
        keywords = {Antimicrobial; peptides;OS-glycopeptides;GlycocinsMicrobial system},
             url = {http://crdd.osdd.net/open/2623/},
        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.}
}