Molecular and genetic insights into the biosynthesis of cyanobacterial neurotoxins

Abstract

Evidence is mounting that the incidence and intensity of harmful algal blooms is increasing, most likely as a result of climate change and its consequential environmental effects. Bloomforming cyanobacteria can produce an array of natural products including toxic secondary metabolites known as cyanotoxins. Among the most significant cyanotoxins from a water management as well as a biomedical perspective are saxitoxin and its chemical analogues, known collectively as the paralytic shellfish toxins (PSTs). This thesis expands our understanding of PST production in cyanobacteria, with a focus on the genetic and molecular elements responsible for the formation of specific PSTs that collectively give rise to unique toxin profiles. Toxic cyanobacteria strains from around Australia were investigated to determine correlations between phenotype, genotype and toxicity. Examination of the toxic strains, belonging to the species Dolichospermum circinale, revealed strain-specific PST profiles. A comparative analysis of these strains revealed that morphology, phylogeny or geography did not appear to be responsible for the variation in toxin quotas. Next generation sequencing was used to analyse and compare the genomes of a subset of these toxic strains to identify genetic elements responsible for PST profile variation. The presence of genes putatively involved in the formation and regulation of PSTs was consistent in all four genomes, as well as a toxic D. circinale reference genome. However, analysis of the gene cluster responsible for the biosynthesis of PSTs (sxt) uncovered tailoring genes previously not reported in any other homologous cluster, and essential for the production of specific PSTs making up the toxin profile of each strain. An expression system was developed for the characterisation of cryptic tailoring enzymes from the sxt gene cluster. Using this system, the sxtX gene, putatively responsible for the conversion of saxitoxin to neosaxitoxin, was expressed as a series of truncated SxtX polypeptides, providing insight into the likely cofactor required for this enzymatic step. The results of this thesis present novel insights into the genetic and molecular factors governing PST production and regulation, expanding our knowledge of these cyanobacterial neurotoxins.