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.
Highlights
Nonribosomal peptides (NRPs) and polyketides (PKs) are two major classes of secondary metabolites with important therapeutic properties, such as antibiotic, antifungal, immunosuppressive, or antitumor activities. They are produced in bacteria and fungi by multimodular enzymatic assembly lines, known as nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), respectively (Finking and Marahiel 2004; Smith and Tsai 2007)
Due to similarities in their modular organization and biosynthetic strategies, PKSs and NRPSs are able to assemble into hybrid multienzyme complexes (Du et al 2001)
The kalimantacin enzymatic assembly line is composed of three polypeptides (Bat1, Bat2, and Bat3), harboring 11 trans-acyltransferase (AT) PKS modules and one NRPS module
Summary
Nonribosomal peptides (NRPs) and polyketides (PKs) are two major classes of secondary metabolites with important therapeutic properties, such as antibiotic, antifungal, immunosuppressive, or antitumor activities They are produced in bacteria and fungi by multimodular enzymatic assembly lines, known as nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), respectively (Finking and Marahiel 2004; Smith and Tsai 2007). The substrate-binding pocket of A domains is lined by 10 AA residues that determine the nature of the AA that is incorporated into the NRP backbone This specificity-conferring code can be bioinformatically extracted and enables the structure prediction of new NRPs based on the genomic arrangement of the corresponding biosynthetic genes (Stachelhaus et al 1999; Challis et al 2000; Röttig et al 2011). The functionality and specificity of the mutated A domains were subsequently analyzed by ultrahigh-resolution LC-MS analysis of culture supernatants
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