title: A subdomain swap strategy for reengineering nonribosomal peptides.
abstract: Nonribosomal peptide synthetases (NRPSs) protect microorganisms from environmental threats by producing diverse siderophores, antibiotics, and other peptide natural products. Their modular molecular structure is also attractive from the standpoint of biosynthetic engineering. Here we evaluate a methodology for swapping module specificities of these mega-enzymes that takes advantage of flavodoxin-like subdomains involved in substrate recognition. Nine subdomains encoding diverse specificities were transplanted into the Phe-specific GrsA initiation module of gramicidin S synthetase. All chimeras could be purified as soluble protein. One construct based on a Val-specific subdomain showed sizable adenylation activity and functioned as a Val-Pro diketopiperazine synthetase upon addition of the proline-specific GrsB1 module. These results suggest that subdomain swapping could be a viable alternative to previous NRPS design approaches targeting binding pockets, domains, or entire modules. The short length of the swapped sequence stretch may facilitate straightforward exploitation of the wealth of existing NRPS modules for combinatorial biosynthesis.
authors.affiliation: Laboratory of Organic Chemistry, ETH Zurich, 8093 Zürich, Switzerland.
authors.forename: Hajo
authors.initials: H
authors.lastname: Kries
authors.affiliation: Laboratory of Organic Chemistry, ETH Zurich, 8093 Zürich, Switzerland.
authors.forename: David L
authors.initials: DL
authors.lastname: Niquille
authors.affiliation: Laboratory of Organic Chemistry, ETH Zurich, 8093 Zürich, Switzerland. Electronic address: hilvert@org.chem.ethz.ch.
authors.forename: Donald
authors.initials: D
authors.lastname: Hilvert
mesh_terms: D000591:Amino Acid Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D013058:Mass Spectrometry; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D010455:Peptides; D011994:Recombinant Proteins; D000591:Amino Acid Isomerases; C022343:phenylalanine racemase (ATP-hydrolyzing); D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase