Rapid screening and evaluation of site saturated structural variants of lantibiotic, nukacin ISK-1

Here we reports the feasibility of introducing saturation mutagenesis (NNK mutagenesis) in nukacin ISK-1 structural gene and to create a large number of structural analogues of this peptide to study the structure function relationship. Three individual residues as Lys1, Pro8 and His12 from the parent peptide were scanned where mutation frequencies were estimated to be around 70% in all cases. A rapid screening method for identification of a large number of nukacin ISK-1 variants form whole cell using a modified MALDI-TOF/MS method have also been developed. As many variants appeared multiple times, therefore after DNA sequencing it could be obtained a total of 37 nukacin ISK-1 variants were obtained for these three positions. We evaluated the potency of each variant by a modified spot on lawn method regardless of their productivity and hence concluded that Lys1 and Pro8 positions are highly variable whereas His12 position is relatively conserved to retain the antimicrobial activity.