- Title
- The distribution, regulation and heterologous expression of cylindrospermopsin: a cyanotoxin with an Australian history
- Creator
- Cullen, Alescia
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2022
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- The formation of cyanobacterial (blue-green algae) blooms in Australian waters are a familiar sight, and of particular concern are Raphidiopsis spp. blooms producing the toxin cylindrospermopsin (CYN). The production of CYNs by Raphidiopsis is, however, not the only cause of concern surrounding the species. Raphidiopsis blooms have been detected on every continent (except Antarctica), with their global distribution due to their invasive nature. To address these risks, this thesis sought to investigate the regulation of the CYN biosynthetic gene cluster (cyr), heterologously produce CYN in E. coli and investigate the specialised metabolite potential of the Raphidiopsis genus. The investigation of specialised metabolite biosynthetic gene clusters (BGCs) from 47 globally diverse Raphidiopsis strains showed strong conservation of BGCs that encode nitrogen fixation, light harvesting, and stress resistance. These metabolites are predicted to contribute to the invasive nature of Raphidiopsis by increasing cellular fitness. On the other hand, BGCs encoding for toxin production and novel non-ribosomal peptides were of lower frequency and their presence correlated strongly to strain geography. Interestingly, despite structural gene rearrangement of the cyr cluster across Raphidiopsis strains, transcriptional regulatory regions within the cyr cluster were highly conserved. These conserved regulatory regions were further explored using DNA-affinity capture assays and showed binding of the transcription factor AbrB to the R. raciborskii AWT205 cyrD promoter region. The transcription factor couples cyr gene transcription to nutrient availability and supports previous studies describing the environmental regulation of CYN production by nitrogen availability. The heterologous production of CYNs in Escherichia coli was also explored as a platform to supply the toxin as an analytical standard. The successful cloning, promoter exchange (to Ptet and PT7), and transcription of the R. raciborskii AWT205 cyr cluster within E. coli BAP1 was demonstrated. Unfortunately, the upregulation of cyr genes did not correspond to CYN production, which was not detected in the cell or media. Interestingly, this disconnect between cyr transcription and CYN production was also observed in R. raciborskii AWT205. These results suggested complex regulation (post-transcriptional, post-translational, or enzyme kinetics) of CYN production. Overall, the results of this study increased our understanding of the mechanisms Raphidiopsis use to control CYN production and takes the first steps towards its sustainable production.
- Subject
- cylindrospermopsin; Raphidiopsis; cyanobacterial; Australia
- Identifier
- http://hdl.handle.net/1959.13/1513851
- Identifier
- uon:56774
- Rights
- Copyright 2022 Alescia Cullen
- Language
- eng
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