Mutagenesis of nisin's leader peptide proline strongly modulates export of precursor nisin.
The lantibiotic nisin is produced by Lactococcus lactis as a precursor peptide comprising a 23 amino acid leader peptide and a 34 amino acid post-translationally modifiable core peptide. We previously demonstrated that the conserved FNLD part of the leader is essential for intracellular enzyme-catalyzed introduction of lanthionines in the core peptide and also for transporter-mediated export, whereas other positions are subject to large mutational freedom. We here demonstrate that, in the absence of the extracellular leader peptidase, NisP, export of precursor nisin via the modification and transporter enzymes, NisBTC, is strongly affected by multiple substitutions of the leader residue at position -2, but not by substitution of positions in the vicinity of this site. Export levels of precursor nisin increased by more than 70% for position -2 mutants Asp, Thr, Ser, Trp, Lys, Val and decreased more than 70% for Cys, His, Met. In a strain with leader peptidase, the Pro-2Lys and Pro-2Asp precursor nisins were less efficiently cleaved by NisP than wild type precursor nisin. Taken together, the wild type precursor nisin with a proline at position -2 allows balanced export and cleavage efficiencies by precursor nisin's transporter and leader peptidase.
Biomade Technology Foundation, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.|Gynecological Oncology, University Medical Center Groningen, 9700 RB, Groningen, The Netherlands.
Annechien
A
Plat
Lanthio Pharma, MorphoSys AG, Rozenburglaan 13B, 9727 DL, Groningen, The Netherlands.
Anneke
A
Kuipers
ImmuCell Corp., 56 Evergreen Drive, Portland, ME, 04130, USA.
Joe
J
Crabb
Lanthio Pharma, MorphoSys AG, Rozenburglaan 13B, 9727 DL, Groningen, The Netherlands.
Rick
R
Rink
Lanthio Pharma, MorphoSys AG, Rozenburglaan 13B, 9727 DL, Groningen, The Netherlands. Moll@lanthiopharma.com.|Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands. Moll@lanthiopharma.com.
Gert N
GN
Moll
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics; D003001:Cloning, Molecular; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics; D016296:Mutagenesis; D009561:Nisin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011392:Proline / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics*; D021381:Protein Transport; D012697:Serine Endopeptidases / Q000235:genetics; D013381:Subtilisins / Q000235:genetics
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D009561:Nisin; D011392:Proline; C080937:NisP protein, Lactococcus lactis; D012697:Serine Endopeptidases; D013381:Subtilisins; C026515:type I signal peptidase
Lactococcus lactis; Lanthionine; Lanthipeptide; Leader peptide; Nisin; Transporter