title: Systematic analysis of the kalimantacin assembly line NRPS module using an adapted targeted mutagenesis approach.
abstract: Kalimantacin is an antimicrobial compound with strong antistaphylococcal activity that is produced by a hybrid trans-acyltransferase polyketide synthase/nonribosomal peptide synthetase system in Pseudomonas fluorescens BCCM_ID9359. We here present a systematic analysis of the substrate specificity of the glycine-incorporating adenylation domain from the kalimantacin biosynthetic assembly line by a targeted mutagenesis approach. The specificity-conferring code was adapted for use in Pseudomonas and mutated adenylation domain active site sequences were introduced in the kalimantacin gene cluster, using a newly adapted ligation independent cloning method. Antimicrobial activity screens and LC-MS analyses revealed that the production of the kalimantacin analogues in the mutated strains was abolished. These results support the idea that further insight in the specificity of downstream domains in nonribosomal peptide synthetases and polyketide synthases is required to efficiently engineer these strains in vivo.
authors.affiliation: Laboratory of Gene Technology, KU Leuven, Kasteelpark Arenberg 21 box 2462, Heverlee, B-3001, Belgium.
authors.forename: Birgit
authors.initials: B
authors.lastname: Uytterhoeven
authors.affiliation: Laboratory of Gene Technology, KU Leuven, Kasteelpark Arenberg 21 box 2462, Heverlee, B-3001, Belgium.
authors.forename: Kenny
authors.initials: K
authors.lastname: Appermans
authors.affiliation: Department of Chemistry, University of Warwick, Gibbet Hill, Coventry, CV4 7AL, United Kingdom.
authors.forename: Lijiang
authors.initials: L
authors.lastname: Song
authors.affiliation: Laboratory of Gene Technology, KU Leuven, Kasteelpark Arenberg 21 box 2462, Heverlee, B-3001, Belgium.|Department of Chemistry, University of Warwick, Gibbet Hill, Coventry, CV4 7AL, United Kingdom.
authors.forename: Joleen
authors.initials: J
authors.lastname: Masschelein
authors.affiliation: Laboratory of Gene Technology, KU Leuven, Kasteelpark Arenberg 21 box 2462, Heverlee, B-3001, Belgium.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lathouwers
authors.affiliation: Centre for Food and Microbial Technology, KU Leuven, Kasteelpark Arenberg 23 box 2457, Heverlee, B-3001, Belgium.
authors.forename: Chris W
authors.initials: CW
authors.lastname: Michiels
authors.affiliation: Laboratory of Gene Technology, KU Leuven, Kasteelpark Arenberg 21 box 2462, Heverlee, B-3001, Belgium.
authors.forename: Rob
authors.initials: R
authors.lastname: Lavigne
mesh_terms: D053898:Biosynthetic Pathways; D002219:Carbamates / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D003062:Codon; D005798:Genes, Bacterial; D013058:Mass Spectrometry; D016296:Mutagenesis; D009154:Mutation; D010453:Peptide Synthases / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D011551:Pseudomonas fluorescens / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D002219:Carbamates; D003062:Codon; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase
keywords: Adenylation domain; kalimantacin; ligation independent cloning; specificity-conferring code