Genome sequencing, analysis, heterologous expression and genetic modification of antibiotic encoding biosynthetic gene clusters

Abstract

Microbial natural products represent an unparalleled starting point for drug discovery. This thesis therefore focuses on microbial natural products, and the biosynthetic gene clusters (BGCs) that encode them. A series of investigations are reported including genome sequencing, genome reading and the exploration of heterologous expression toward enabling the discovery of novel natural products. The heterologous expression of large and highly repetitive BGCs was investigated, in addition to the heterologous expression of individual cytochrome P450 enzymes to complement existing biosynthetic pathways, as an approach toward accessing new to nature natural products.

Firstly, a series of novel BGCs were identified in silico from public databases and from novel marine strains which were sequenced using MinION nanopore technology. The rationale being that novel BGCs are likely to encode for new compounds. BGCs encoded for by Saccharothrix espanaensis were heterologously expressed and molecular networking of mass spectrometry data helped to identify masses uniquely produced by the heterologously expressed BGCs.

Next, the putative BGC encoding for Marinomycin A, a potent antibiotic and anticancer agent produced by Marinispora CNQ-140, was also heterologously expressed using a direct-cloning approach. Future work will involve both promoter refactoring and sequencing of the cloned BGC to identify any sequence rearrangements, as Marinomycin A production could not be detected in the heterologous host. Moreover, the construction of a Marinispora CNQ-140 BAC library, a library containing an average insert size of 145 kb, represents an alternate way to capture and heterologously express this 72 kb BGC, with its challengingly high level of sequence repetition. Genome sequencing of Marinispora CNQ-140 also revealed additional novel BGCs which can be targeted using this BAC library.

Lastly, work towards establishing a biocatalyst toolkit to enable the epoxidation of a series of natural products was conducted. An introduced epoxide can act as a biorthogonal handle, therefore facilitating downstream chemical modifications of the natural product. Success was gained using a series of evolved P450 BM3 variants in vitro, which were able to convert substrates such as Novobiocin and Pimaricin. Moreover, a series of cytochrome P450s from uncharacterised BGCs with origins similar to a series of compounds that we wished to modify, were also mined and tested.