Unveiling the iterative programming of the zeamine II enzymatic assembly line Sofie Dekimpe 1,2, Valentin Colpaert 1 , Ruben De Roeve 1 , Loes Temmerman 1 , Matthew Jenner 3 , Rob Lavigne 1 , Joleen Masschelein 2 1 KU Leuven, Belgium, 2 Laboratory for Biomolecular Discovery and Engineering, KU Leuven, Belgium, 3 University of Warwick, UK The zeamines are an unusual class of specialized metabolites produced by Gram-negative Serratia spp. 1 They display antagonistic activity against a wide range of organisms, including bacteria, nematodes, plants and fungi. This broad spectrum bioactivity is linked to the cationic amphiphilic nature of the zeamines and their ability to directly interact with and disrupt the integrity of cellular membranes. 2 The zeamine antibiotic complex is a mixture of five structurally-related compounds, all of which contain a 40-carbon pentaamino-hydroxyalkyl chain connected to a variable peptide-polyketide moiety. Zeamine assembly is directed by a unique combination of nonribosomal peptide (NRP), polyketide (PK) and polyunsaturated fatty acid (PUFA) biosynthetic machinery. The biosynthetic pathway involves two distinct enzymatic assembly lines that operate in parallel. In the first pathway, an iterative type I PKS/PUFA synthase-like multienzyme complex forms the 40-carbon polyamino alcohol chain zeamine II. In the second pathway, a hybrid NRPS/PKS assembles a hexapeptide mono/diketide moiety. Both products are linked via intermolecular condensation, catalysed by a stand-alone condensation domain. 1,3 In this study, we aimed to decipher the biosynthetic logic of zeamine II assembly. The zeamine II pathway has likely evolved from PUFA synthases and has acquired additional functionalities, such as a cis -acting aminotransferase, ketoreductase and thioester reductase domain (see figure below). Using a combination of in vivo mutagenesis, in vitro enzymatic studies, chemical synthesis and MS 2 Ppant ejection assays, we functionally characterized the distinct biosynthetic domains and elucidated their substrate selectivity. Moreover, we reconstituted the distinct steps of the zeamine II assembly in vitro , providing important insights into the iterative programming of this unusual assembly line. Our findings open up new opportunities for rational engineering of the zeamine pathway for the production of novel antibiotic analogues with improved pharmacological properties.
Figure 1. The zeamine II assembly line. References 1. Masschelein, J. et al. A PKS/NRPS/FAS Hybrid Gene Cluster from Serratia plymuthica RVH1 Encoding the Biosynthesis of Three Broad Spectrum, Zeamine-Related Antibiotics. PLoS One 8 , e54143 (2013). 2. Masschelein, J. et al. The zeamine antibiotics affect the integrity of bacterial membranes. Appl. Environ. Microbiol. 81 , 1139–1146 (2015). 3. Masschelein, J. et al. A combination of polyunsaturated fatty acid, nonribosomal peptide and polyketide biosynthetic machinery is used to assemble the zeamine antibiotics. Chem. Sci. 6 , 923–929 (2015).
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