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-PPi 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.
