Building and analysing synthetic biological and GenoChemetic systems for accessing natural products and their analogues

Abstract

This thesis discusses a variety of synthetic biological approaches to advance the tools for the sustainable production and biosynthetic modification of natural products. Firstly, it outlines a pipeline for the discovery of cytochrome P450 epoxidases from genomic information. By building on work carried out by previous Goss group members, a series of known and putative cytochrome P540 epoxidases were assessed for activity against clinically relevant polyenes. These target enzymes were heterologously expressed and the bioreactions with the chosen substrates explored. These samples were assessed for substrate conversion and target product generation in a selection of heterologous hosts. Based on this, the most promising enzyme-host system for each substrate could be selected.

It was also necessary to assess the potential for product generation at levels beyond an analytical scale and so scale-up conditions were considered. During this phase, protocols for purification of the epoxy-product were developed and product was purified.

In addition to individual gene identification encoding cytochrome P450 epoxidases, the identification of two biosynthetic gene clusters was achieved through in silico experiments and in vitro exploration. Firstly, the BGC for marinomycin was heterologously expressed in a Streptomyces sp. host strain. The BGC for marinomycin is a large highly repetitive PKS cluster and so presented unique challenges, as recombination was highly likely when pursuing traditional cloning methods. The high levels of genetic repetition was investigated via bioinformatic analysis. By heterologous expression of a member of a BAC library, the production was enabled and the product was detected in negative mode via LC-MS analysis. Additionally, the BGC for another antibiotic, vD844, was identified and steps were taken towards the generation of a heterologous expression system. vD844 Was an interesting candidate for heterologous expression due to a chemically orthogonal handle, specifically an N-formyl group, being biosynthetically produced. Plasmids containing the whole BGC were generated from a cosmid library. Subsequent to the completion of work within this thesis, the heterologous production has been confirmed.