Elucidating and engineering bottromycin biosynthesis

Bottromycin is a structurally unique peptide that possesses potent antibacterial activity towards life-threatening infections, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). The structural novelty of bottromycin stems from a macrocyclic amidine and Beta-methylated amino acids. Novel classes of antimicrobials are rare, so these features make bottromycin an attractive molecule for both biosynthetic and bioactivity analysis. I have previously identified the biosynthetic gene cluster for bottromycin in Streptomyces scabies, a pathogen that is the causative agent of potato scab and demonstrated that bottromycin derives from a ribosomal peptide. The overall goal of this research programme is to employ complementary genetic and biochemical methods to elucidate the mechanisms of these unknown biosynthetic steps. The characterisation of the unprecedented amidine-forming macrocyclisation is the main priority. Novel bottromycin-like molecules will be generated by pathway engineering and by employing the cyclase as a biocatalyst.

The first objective is to identify the enzymes that catalyse macrocyclisation and thiazoline formation.?In vitro and in vivo methods will be employed to identify the amidine-forming cyclase and the heterocyclase responsible for thiazoline formation. Objective two involves generate bottromycin analogues and probing the substrate tolerance of the pathway by using the amidine-forming cyclase as a biocatalyst, as well as by using site-directed mutagenesis to generate bottromycin analogues in vivo.
The third objective is to elucidate the regulatory basis of bottromycin biosynthesis. The precise role of the one pathway-specific regulatory protein will be probed by DNA-binding assays. Objective four will focus on reengineering the regulation of the pathway to increase the quantity of bottromycin analogues for bioactivity analysis.