Generation of a library of recombineered novel polyketides and non-ribosomal peptides

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Polyketide natural products made by microorganisms such as bacteria and fungi represent a major source of antibiotic and anticancer pharmaceuticals for the treatment of life threatening disease. They are also important as immunosuppressant agents after transplant surgery, as drugs for the treatment of parasitic diseases in developing countries and as environmentally benign insecticides for use in crop protection. Despite this historical success polyketides are underutilised for the discovery of new medicines, in part due to the perceived lack of a repeatable and scalable process for their development that can compete with other technologies used by the modern pharmaceutical and agrochemical industries.This project will aid our industrial partner Isomerase in the development of new genetic methods to modify bacterial machinery (enzymes) that make polyketide natural products, and provide us with a deeper understanding of the processes leading to their natural evolution. These enzymes act as assembly lines with an individual 'part' or 'domain' to perform each chemical step in the assembly process. In order to produce a complete polyketide these domains are assembled in repeating modules which use simple precursors from the cell, bond these together and then modify them in order to generate the complex final molecule. Our proposal will build on recent advances in DNA sequencing which enables long, repetitive stretches of DNA to be sequenced accurately and quickly. This is important for the long and repetitive regions of DNA making up the genes that encode polyketide synthase enzymes which include some of the largest proteins known to nature. We will sequence the genomes of 10 strains generated through early application of Isomerase's 'recombineering' technology. Amazingly these produce a structurally diverse library of compounds based on a single nautural structure and were derived from just a single experiment. We will analyse the recombineered genes in order to identify the recombination hotspots and derive an understanding of natural and induced examples of recombineering This will also aid us in devising better rational experiments for making discrete changes to polyketide synthases in order to produce specific targeted compounds.The compounds produced by this and subsequent experiments have the potential to be leads for the discovery of new medicines including anti-infective agents for the treatment of drug resistant bacteria and emerging viruses which represent immediate and alarming public health threats.