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

Abstract

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 Lactococcus lactis, 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.

• Authors: Xinghong Zhao, Rubén Cebrián, Yuxin Fu, Rick Rink, Tjibbe Bosma, Gert N Moll, Oscar P Kuipers
• Journal: ACS synthetic biology (ACS Synth Biol) Vol. 9 Issue 6 Pg. 1468-1478 (06 19 2020) ISSN: 2161-5063 [Electronic] United States
• PMID: 32374981 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Bacterial Proteins
• Ligands
• Membrane Proteins
• NisB protein, Lactococcus lactis
• Peptides
• Sulfides
• Streptavidin
• lanthionine
• Alanine

Topics

• Alanine (analogs & derivatives, chemistry)
• Amino Acid Sequence
• Bacterial Proteins (genetics, metabolism)
• High-Throughput Screening Assays (methods)
• Lactococcus lactis (enzymology)
• Ligands
• Membrane Proteins (genetics, metabolism)
• Mutagenesis, Site-Directed
• Peptides (chemistry, metabolism)
• Protein Binding
• Streptavidin (chemistry, metabolism)
• Substrate Specificity
• Sulfides (chemistry)