Systematic analysis of the kalimantacin assembly line NRPS module using an adapted targeted mutagenesis approach. Issue 2 (15th December 2015)

• Record Type: Journal Article
• Authors: Uytterhoeven, Birgit; Appermans, Kenny; Song, Lijiang; Masschelein, Joleen; Lathouwers, Thomas; Michiels, Chris W.; Lavigne, Rob
• Is Part Of: MicrobiologyOpen. Volume 5:Issue 2(2016:Apr.)
• Page Start: 279
• Page End: 286
• Publication Date: 2015-12-15
• Subjects: Adenylation domain -- kalimantacin -- ligation independent cloning -- specificity‐conferring code; Microbiology -- Periodicals; 579
• DOI: 10.1002/mbo3.326
• Languages: English
• ISSNs: 2045-8827

Abstract

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 . Abstract : In this study, the specificity of the glycine‐incorporating adenylation domain of the nonribosomal peptide synthetases (NRPS) module in the kalimantacin biosynthetic assembly line is adapted by use of a Pseudomonas‐specific specificity‐conferring code. A very efficient ligation independent cloning method is presented to replace the active site sequence of the adenylation domain in the parent strain with a chemically synthesized DNA fragment with the intended mutations. This study emphasizes the need for fundamental knowledge in the substrate specificity of the different domains in hybrid PKS/NRPS clusters.