High-Throughput Screening for Substrate Specificity-Adapted Mutants of the Nisin Dehydratase NisB
| Autor: | Rick Rink, Tjibbe Bosma, Rubén Cebrián, Xinghong Zhao, Gert N. Moll, Oscar P. Kuipers, Yuxin Fu |
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| Přispěvatelé: | Molecular Genetics |
| Rok vydání: | 2020 |
| Předmět: |
0106 biological sciences
Streptavidin DIRECTED EVOLUTION Biomedical Engineering Sulfides high-throughput screening 01 natural sciences Biochemistry Genetics and Molecular Biology (miscellaneous) Substrate Specificity 03 medical and health sciences chemistry.chemical_compound dehydratase Bacterial Proteins DESIGN Dehydroalanine 010608 biotechnology Amino Acid Sequence Nisin Lanthionine 030304 developmental biology 0303 health sciences Bacterial display Alanine biology NisB Lactococcus lactis Membrane Proteins PEPTIDES General Medicine Directed evolution biology.organism_classification High-Throughput Screening Assays SURFACE DISPLAY Biochemistry chemistry DISCOVERY Dehydratase STREPTAVIDIN Mutagenesis Site-Directed LIGANDS bacterial display Research Article Protein Binding |
| Zdroj: | ACS Synthetic Biology, 9(6), 1468-1478. AMER CHEMICAL SOC ACS Synthetic Biology |
| ISSN: | 2161-5063 |
| DOI: | 10.1021/acssynbio.0c00130 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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