Open AccessSystematic analysis of the kalimantacin assembly line NRPS module using an adapted targeted mutagenesis approach
Author(s) -
Uytterhoeven Birgit,
Appermans Kenny,
Song Lijiang,
Masschelein Joleen,
Lathouwers Thomas,
Michiels Chris W.,
Lavigne Rob
Publication year - 2016
Publication title -
microbiologyopen
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.881
H-Index - 36
ISSN - 2045-8827
DOI - 10.1002/mbo3.326
Subject(s) - nonribosomal peptide , adenylylation , polyketide , polyketide synthase , mutagenesis , pseudomonas fluorescens , biology , gene cluster , biochemistry , dna ligase , site directed mutagenesis , mutant , gene , computational biology , genetics , biosynthesis , bacteria
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 _ ID 9359. 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 .