Unnatural production of natural products: Heterologous expression and combinatorial biosynthesis of novel cyanobacterial-derived compounds

Cyanobacteria produce a vast array of structurally unique secondary metabolites with useful bioactive properties. These compounds are often expressed by multi-modular nonribosomal peptide synthetases (NRPS). Heterologous expression of a variety of microbial natural compounds has been used to harness their diversity and to facilitate their combinatorial biosynthesis. However, these genetic techniques have not been developed for secondary metabolite-producing cyanobacterial species. Therefore the genetically manipulable cyanobacterium Synechocystis sp. PCC6803 was engineered for NRPS compound production. Firstly, the phosphopantetheinyl transferase (PPT) from this species, Sppt, was characterised in order to determine its ability to activate NRPS carrier proteins from secondary metabolite pathways. Despite bioinformatic analyses which suggested Sppt has a broad substrate specificity, phosphopantetheinylation assays and enzyme kinetics revealed it is a dedicated PPT for fatty acid synthesis. Consequently, the broad-range PPT from the filamentous cyanobacteria Nodularia spumigena NSOR10 was chromosomally integrated into this strain, which allowed it to activate the biosynthetic machinery involved in nonribosomal peptide synthesis. Cyanobacterial natural product engineering was also explored with the characterisation of two relaxed specificity A-domains from the biosynthetic pathway of the toxin microcystin. The wide variety of microcystin compounds produced by cyanobacterial species suggests that multiple amino acid substrates can be activated by the same A-domain. This was confirmed using ATP-PP_i exchange assays and was subsequently harnessed by the in vitro production of a variety of dipeptides using an engineered relaxed dimodule. Therefore, this study provides a biotechnological platform for the heterologous expression of novel cyanobacterial-derived compounds.