Elucidating and exploiting halogenase recruitment beta hairpin docking domains in nonribosomal peptide biosynthesis

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Nonribosomal peptides and polyketides are classes of natural products, comprising a large variety of biologically-relevant compounds. They are synthesised by nonribosomal peptide synthases (NRPSs) and polyketide synthases (PKSs) respectively, which are modular megasynthases that act in an assembly line-like manner. The interaction between subunits of PKS and NRPS proteins is critical to the fidelity of their biosyntheses, and are known in many cases to be facilitated by C-terminal and N-terminal docking domains. One type of interaction between subunits in hybrid NRPS-PKS systems involves the docking of -hairpin docking domains (HDDs) attached to catalytic domains at the N-terminus of one subunit with a short linear motifs (SLiMs) attached to carrier proteins at the C-terminus of another subunit. Recent computational work in the Challis group has suggested that this system of docking domains may be far more prevalent in the biosynthetic machinery of polyketide and nonribosomal peptide metabolites than first appreciated. This project will focus upon the characterisation of HDD/SLiM interactions predicted to occur in the biosynthetic pathway of nonribosomal peptides from cyanobacteria. The aims of the project are to experimentally characterise the HDD/SLiM interactions by overproducing the relevant proteins in E. coli and using synthetic substrate analogues to examine their enzymatic activity. We will also attempt to obtain high resolution structural information for the proteins using X-ray crystallography and NMR spectroscopy. Once characterised, this system will be exploited to understand the potential of the HDD/SLiM systems as a tool for engineering of biosynthetic pathways, including the production of hybrid assembly lines using components from the biosyntheses of different metabolites.