Molecular Investigation of Inter-Adenylation Domain Consensus Primers to Aid in the Elucidation of Cyanobacterial Non-Ribosomal Peptide Synthetase Gene Clusters

Abstract

Non-ribosomal peptide synthetases (NRPSs) are highly refined natural machines for the biosynthesis of secondary oligopeptides which cannot be synthesised by the ribosomal machinery. Due to the attractive biological potential of many of these secondary metabolites, like anticancer, antimicrobial, antiviral, toxic, and immunosuppressive activities etc, a great interest to investigate these systems for further exploitation and understanding have taken place in recent years. Because of a relative linear modular structure of NRPSs, they have become potential targets for combinatorial manipulations to reprogram the natural peptides to aid in the discovery of improved and more active peptides

Cyanobacteria are a rich source of these secondary metabolites and found to be one of the oldest living organisms which have developed these biosynthetic pathways. Genetic elucidation of these pathways has been described by a large body of literature for the past two decades, but yet there are only a few completely described biosynthetic pathways. So far the majority of the genetic data available is based on intra-domainal sequences of the adenylation domain within these NRPS gene clusters. As these sequences might only reveal the residues being activated, they cannot give any information about which gene clusters are present, or their pathway towards the final oligopeptide. To aid in the elucidation of these pathways, investigations have been conducted to use inter-adenylation domainal consensus primers to easily obtain the whole gene cluster with simple molecular techniques. These analyses have demonstrated that it is possible to use such consensus primers to elucidate NRPS genes. Combining intra-adenylation and inter-adenylation fragments, sequence data containing up to 8600 bp have been obtained. Unfortunately due to some template contamination, it was not possible to elucidate from which cyanobacterial strain these sequences might originate from.