High-Throughput Screening for Substrate Specificity-Adapted Mutants of the Nisin Dehydratase NisB
Xinghong Zhao, Rubén Cebrián, Yuxin Fu, Rick Rink, Tjibbe Bosma, Gert N. Moll, Oscar P. Kuipers
Abstract
, a cell surface display precursor was designed comprising DSHPQFC. The Asp residue preceding the serine in this sequence disfavors its dehydration by wild-type NisB. The cell surface display vector was coexpressed with a mutant NisB library and NisTC. Subsequently, mutant NisB-containing bacteria that display cyclized strep ligands on the cell surface were selected via panning rounds with streptavidin-coupled magnetic beads. In this way, a NisB variant with a tailored capacity of dehydration was obtained, which was further evaluated with respect to its capacity to dehydrate nisin mutants. These results demonstrate a powerful method for selecting lanthipeptide modification enzymes with adapted substrate specificity.