High-throughput screening for substrate specificity-adapted mutants of the nisin dehydratase NisB

Zhao, X., Cebrian, R., Fu, Y., Rink, R., Bosma, T., Moll, G. N. & Kuipers, O. P., 2020, In : ACS Synthetic Biology.

Research output: Contribution to journalArticleAcademicpeer-review

Microbial lanthipeptides are formed by a two-step enzymatic introduction of (methyl)lanthionine rings. A dehydratase catalyzes the dehydration of serines/threonines, yielding dehydro-alanine and dehydro-butyrine, respectively. Cyclase-catalyzed coupling of the formed dehydro-residues to cysteines forms (methyl)lanthionine rings in a peptide. Lanthipeptide biosynthetic systems allow to discover target-specific, lanthionine-stabilized therapeutic peptides. However, the substrate specificity of existing modification enzymes impose limitations in installing lanthionines in non-natural substrates. Goal of the present study was to obtain a lanthipeptide dehydratase with the capacity to dehydrate substrates that are unsuitable for the nisin dehydratase NisB. We report high-throughput screening for tailored specificity of intracellular, genetically encoded NisB dehydratases. The principle is based on the screening of bacterially-displayed lanthionine-constrained streptavidin ligands, that have a much higher affinity for streptavidin than linear ligands. The designed, NisC-cyclizable high-affinity ligands can be formed via mutant NisB-catalyzed dehydration, but less effectively via wild type NisB activity. In Lactococcus lactis, a cell surface display precursor was designed, comprising DSHPQFC. The preceding Asp residue of the serine in this sequence disfavors its dehydration by wild type NisB. The cell surface display vector was co-expressed 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 dehydrated nisin mutants. These results demonstrate a powerful method for selecting lanthipeptide modification enzymes with adapted substrate specificity.

Original languageEnglish
JournalACS Synthetic Biology
Publication statusE-pub ahead of print - 2020

ID: 124744566