SELECT-GLYCOCIN: a recombinant microbial system for expression and high-throughput screening of glycocins

• Published in: Glycoconjugate journal 2021-04, Vol.38 (2), p.233-250
• Main Authors: Choudhary, Pravinkumar, Rao, Alka
• Format: Article
• Language: English
• Subjects: Anti-Bacterial Agents - pharmacology; Antibiotics; Antimicrobial agents; Bacteriocins; Bacteriocins - genetics; Bacteriocins - metabolism; Bacteriocins - pharmacology; Biochemistry; Biological activity; Biomedical and Life Sciences; Enterococcus faecalis - genetics; Escherichia coli - genetics; Food; Glycopeptides; Glycosylation; Glycosyltransferase; Glycosyltransferases - genetics; Glycosyltransferases - metabolism; Hemolysis - drug effects; High-throughput screening; High-Throughput Screening Assays - methods; Humans; Life Sciences; Listeria monocytogenes - drug effects; Microbial Sensitivity Tests; Mutants; Mutation; Original Article; Pathology; Peptide Library; Preservatives; Proof of Concept Study; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Toxicity; Young Investigator Special Issue
• ISSN: 0282-0080; 1573-4986
• DOI: 10.1007/s10719-020-09960-w

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.