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

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.

Bioengineering is complementary to semi-synthesis for the structural diversification and lead optimization of biologically active natural products. The ability to bioengineer modular polyketide synthases (PKSs) is of particular interest and has focused on rational approaches to yield focussed libraries. The design rules for modifying modular PKSs retain a significant empirical element and the successful reports have described compounds with modest structural variation and a range of yields.

Our industrial partner Isomerase has identified techniques that induce modular rapamycin PKS genes in Streptomyces rapamycinicus to undergo a 'recombinatorial' process leading, in a single experiment, to multiple progeny each encoding functional contracted or expanded PKSs producing new rapalogs in good to excellent yield.

We will sequence 9 of these strains, plus parent, using PacBio sequencing which we recently used to give single contig genomes for several Streptomyces & Pseudomonas strains in collaboration with TGAC. Multimodular PKS & NRPS sequences (of very high sequence similarity) are perfectly assembled using this method which makes it ideal for sequencing the rapamycin PKS progeny, a problem for other methods. Using detailed bioinformatic analysis we will identify the recombination boundaries and and associated physical parameters (minimum sequence length, module location etc.). This will aid further understanding of the recombinatorial technique in order to to generate compound diversity for other systems as well as providing more refined guiding principles for rational PKS bioengineering.

Ultimately, we aim to establish a synthetic biology platform where any modular PKS or NRPS genes can be synthesised and expressed in a heterologous host enabled for induced recombination. When combined with selection methods this would provide an unparralleled new approach for the discovery of natural products based pharmaceutical, agrochemcials & high value chemicals

As described in proposal submitted to TSB