SELECT-GLYCOCIN: a recombinant microbial system for expression and high-throughput screening of glycocins
- PDF / 691,525 Bytes
- 18 Pages / 595.276 x 790.866 pts Page_size
- 76 Downloads / 144 Views
ORIGINAL ARTICLE
SELECT-GLYCOCIN: a recombinant microbial system for expression and high-throughput screening of glycocins Pravinkumar Choudhary 1 & Alka Rao 1,2 Received: 3 August 2020 / Revised: 3 August 2020 / Accepted: 21 October 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
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. Keywords Antimicrobial peptides . O/S-glycopeptides . Glycocins . Microbial system
Introduction Antimicrobial peptides (AMPs) are synthesized by all living organisms and secreted in the surrounding environment to kill other microbes. As such, AMPs exhibit broad-spectrum activities against bacteria, yeast, fungi, and viruses, along with immune-modulatory, anti-inflammatory, and cytotoxic activities against cancer cells. AMPs synthesized by bacteria are termed bacteriocins that provide a competitive advantage for the survival of the bacteria in a given ecological niche [1]. Broadly, bacteriocins could be either unmodified or post* Alka Rao [email protected] 1
CSIR-Institute of Microbial Technology, Sector 39A, Chandigarh 160036, India
2
Academy of Scientific and Innovation Research (AcSIR), Ghaziabad 201002, India
translationally modified, such as thiopeptides, lipolanthines, lasso peptides, and glycosylated bacteriocins (glycocins). Among these, glycocins represent the most recent discovery. As of now, 12 such glycocins are characterized experimentally and listed in Supplementary Fig. S1 and Table 1.
Data Loading...
