Biochemical Dissection of the Natural Diversification of Microcystin Provides Lessons for Synthetic Biology of NRPS.
The cyanobacterial hepatotoxin microcystin is assembled at a non-ribosomal peptide synthetase (NRPS) complex. The enormous structural diversity of this peptide, which is also found in closely related strains, is the result of frequent recombination events and point mutations. Here, we have compared the in vitro activation profiles of related monospecific and multispecific modules that either strictly incorporate leucine or arginine or incorporate chemically diverse amino acids in parallel into microcystin. By analyzing di- and tri-domain proteins we have dissected the role of adenylation and condensation domains for substrate specificity. We have further analyzed the role of subdomains and provide evidence for an extended gatekeeping function for the condensation domains of multispecific modules. By reproducing natural point mutations, we could convert a monospecific module into a multispecific module. Our findings may inspire novel synthetic biology approaches and demonstrate how recombination platforms of NRPSs have developed in nature.
Department of Microbiology, Institute for Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Street 24/25, 14476 Potsdam-Golm, Germany.
Sabine
S
Meyer
Department of Microbiology, Institute for Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Street 24/25, 14476 Potsdam-Golm, Germany.
Jan-Christoph
JC
Kehr
Institut für Chemie, Technische Universität Berlin, Strasse des 17. Juni 124, 10623 Berlin, Germany.
Andi
A
Mainz
Department of Microbiology, Institute for Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Street 24/25, 14476 Potsdam-Golm, Germany.
Daniel
D
Dehm
Institut für Chemie, Technische Universität Berlin, Strasse des 17. Juni 124, 10623 Berlin, Germany.
Daniel
D
Petras
Institut für Chemie, Technische Universität Berlin, Strasse des 17. Juni 124, 10623 Berlin, Germany.
Roderich D
RD
Süssmuth
Department of Microbiology, Institute for Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Street 24/25, 14476 Potsdam-Golm, Germany. Electronic address: editt@uni-potsdam.de.
Elke
E
Dittmann
D052998:Microcystins / Q000737:chemistry / Q000378:metabolism*; D046931:Microcystis / Q000201:enzymology*; D008968:Molecular Conformation; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*
D052998:Microcystins; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase
adenylation domain; condensation domain; cyanobacteria; evolution; multispecificity; non-ribosomal peptide; synthetic biology