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.

糖基化细菌素（甘球菌素）是潜在的清洁标签食品防腐剂和抗生素的新替代品。进一步的发展需要一种糖蛋白实验室进化方法的可用性，其中要克服的挑战包括确保异源宿主中的糖基化，避免活性糖蛋白对宿主的潜在毒性，以及为活性突变体提供一锅筛选试验。采用 EntS，一种来自粪肠球菌TX0104的顺序 O/S-二糖基转移酶，一种概念证明微生物系统和高通量筛选测定 (SELECT-GLYCOCIN) 被开发用于生成 O/S 连接的糖肽文库和筛选糖蛋白所需的活动/财产。该方法能够在体内实现酶依赖性异源宿主中的糖基化和肠霉素 96 (Ent96) 突变体的快速筛选 - 一种对食源性病原体L. 单核细胞增多症有活性的糖蛋白. 使用 SELECT-GLYCOCIN，生成了 Ent96 的随机 (1.5 X 10^3)​​ 和有理 (17) 突变体库。对突变体进行生物活性筛选，以确定总共 376 个随机突变体和 14 个合理突变体具有生物活性。对 16 个随机突变体和 14 个合理突变体的下游详细分析导致鉴定了序列或糖变体，即 G16E-H24Q、C13T 和 Ent96-K4_K5insYYGNGV (PedioEnt96) 作为改进的抗菌剂。总之，SELECT-GLYCOCIN 提供了一种用于发现和筛选甘氨酸的系统和通用方法，可以进一步适应任何已知/未知的甘氨酸，并可用于食品防腐剂和药物发现计划。