Gifts from nature : genomic and metabolomic approaches to natural product discovery from cyanobacteria and actinomycetes

Abstract

Cyanobacteria represent a treasure trove of uncovered natural products (NPs) and unbeknown biosynthetic machinery. Currently the exponential rise in genome sequencing of cyanobacteria and other organisms has revealed a wealth of biosynthetic gene clusters, many of which cannot be linked to a NP.

This work describes the investigation of cyanobacterial strains for the presence of non-ribosomal peptide synthase (NRPS) and polyketide synthase (PKS) genes screened by PCR and Sanger sequencing. In addition to whole genome sequencing (WGS) using Illumina and Oxford Nanopore was undertaken of selected strains. Bioinformatic tools were used to detect biosynthetic gene clusters (BGCs) and identify regions of interest. Furthermore, publicly available data, in the form of publications and nucleotide data from genome assemblies was gathered to form several datasets to analyse and link cyanobacterial metabolites to their genomes, and to uncover the diversity of cyanobacterial NPs.

Recent development has been undergoing to establish heterologous expression tools for cyanobacteria. Currently only a dozen cyanobacterial natural products have been heterologously expressed, this work details the heterologous expression of a ribosomally synthesised and post-translationally modified peptide (RiPP) named viridisamide A from Oscillatoria nigro-viridis PCC 7112 using the DiPaC method. This thesis also describes the use of this method for the cloning of the Fischerazole BGC from Fischerella sp. 9431.

Furthermore, the NP cyclomarin A, a marine natural product from an actinomycetes rather than a cyanobacterium, which possesses potent bioactivities against both tuberculosis and malaria was investigated. Here precursor directed biosynthesis of the fermented strain Streptomyces sp. BCC41611 was used to created halogenated cyclomarin variants. In addition, synthetic chemistry methods were used to functionalise the epoxide of the biosynthesised NP by azidolysis and copper(I) catalysed alkyne-azide cycloaddition.

Lastly, the halogenase VirX1 from the cyanophage syn10 was studied. This phage infects the marine cyanobacterial genera Synechococcus and Prochlorococcus which are responsible for over a quarter of global photosynthesis. Here, the halogenase was investigated in order to attempt to uncover its natural substrates.