High-Throughput Screening for Substrate Specificity-Adapted Mutants of the Nisin Dehydratase NisB

• Published in: ACS synthetic biology Vol. 9; no. 6; pp. 1468 - 1478
• Main Authors: Zhao, Xinghong, Cebrián, Rubén, Fu, Yuxin, Rink, Rick, Bosma, Tjibbe, Moll, Gert N, Kuipers, Oscar P
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 19-06-2020
• Subjects: Alanine - analogs & derivatives; Alanine - chemistry; Amino Acid Sequence; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; High-Throughput Screening Assays - methods; Lactococcus lactis - enzymology; Ligands; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mutagenesis, Site-Directed; Peptides - chemistry; Peptides - metabolism; Protein Binding; Streptavidin - chemistry; Streptavidin - metabolism; Substrate Specificity; Sulfides - chemistry; Lactococcus lactis; streptavidin; bacterial display; high-throughput screening; NisB; dehydratase
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.0c00130

Microbial lanthipeptides are formed by a two-step enzymatic introduction of (methyl)lanthionine rings. A dehydratase catalyzes the dehydration of serine and threonine residues, yielding dehydroalanine and dehydrobutyrine, respectively. Cyclase-catalyzed coupling of the formed dehydroresidues to cysteines forms (methyl)lanthionine rings in a peptide. Lanthipeptide biosynthetic systems allow discovery of target-specific, lanthionine-stabilized therapeutic peptides. However, the substrate specificity of existing modification enzymes impose limitations on installing lanthionines in non-natural substrates. The 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, which 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
, 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.