Molecular analysis of bromoform biosynthesis in the red seaweed Asparagopsis taxiformis

Jessica Webb

doi:10.25907/01025

The red seaweed Asparagopsis taxiformis is a climate-positive feed additive for ruminant livestock due to its ability to reduce enteric methane emissions. As a result, A. Taxiformis is the focus of a developing aquaculture industry to support the large-scale production of biomass. The antimethanogenic effect of the seaweed is due to a high concentration of halogenated bioactive compounds, primarily bromoform, which are synthesised by a group of enzymes known as vanadium bromoperoxidases (VBPOs). However, research is needed to form a better understanding of halogenated metabolite pathways, including identifying the molecular factors involved in bromoform biosynthesis, storage, transport, and post-transcriptional regulation. Over four research chapters, this thesis investigated conditions which influence halogenation to identify bromoform-associated genes, while establishing novel molecular resources for red seaweed biology more broadly. In Chapter 2, a review of VBPO research in seaweeds was conducted, identifying significant knowledge gaps relating to gene regulation, protein localisation, and physiological roles, especially in stress responses. An integrated data analysis presented the most comprehensive list of seaweed VBPOs to date through genome mining of annotated seaweed genomes. Reviewing gene expression analyses revealed that VBPOs are commonly upregulated in response to biotic and chemical stresses, suggesting a conserved role in environmental adaptation. Investigation of gene organisation indicated that the biosynthetic locus described for A. taxiformis is not broadly conserved across seaweeds, suggesting species-specific genomic architectures for halogenation pathways. Chapter 3 established that management of light intensity is a key factor for ptimising both biomass production and bromoform accumulation in A. taxiformis. Exposure to high light at 100 μmol photons m−2 s−1 doubled both growth rate and bromoform concentration relative to 25 μmol photons (photosynthetically active radiation) m−2 s−1. Transcriptomic analyses revealed that, despite increased bromoform levels up to 1.54% dry weight, the two most highly expressed VBPOs were in fact downregulated under the high light conditions. This finding demonstrates that halogenated metabolite production is not driven solely by increased VBPO abundance, highlighting the need for further research surrounding the regulatory mechanisms influencing bromoform biosynthesis. Additionally, genes associated with oxidative stress responses, photosynthesis, and light-responsive transcription factors were identified as highly responsive to changes in light levels, further indicating a complex molecular adaptation to light intensity in this seaweed. Chapter 4 provided the first characterisation of the miRNA landscape in A. taxiformis. Homologues of core miRNA biogenesis genes were identified, demonstrating shared features with plant, animal, and microalgae pathways. Small RNA sequencing identified 16 known miRNA families and 283 novel miRNA families, with 34 novel miRNAs families consistently detected across samples. A. taxiformis miRNAs were found to be predominantly 19 nucleotides in length, shorter than typical plant or animal miRNAs. miRNA target gene prediction indicated that A. taxiformis miRNAs are involved in the regulation of development and stress responses, with several transcription factors and two non-functional vanadium haloperoxidases predicted as target genes. Chapter 5 demonstrated that availability of bromide is essential for normal gland cell development and halogenated compound accumulation. Tetrasporophytes cultured in bromide-free artificial seawater lacked gland cells in new growth, while bromoform concentration decreased by 60%. Transcriptomic analyses proposed specific roles for individual VBPOs, while identifying several bromide-dependent genes with putative involvement in halogenation-related processes, including a transporter, embraneassociated genes, and specific vanadium haloperoxidases. Additionally, a large range of novel miRNAs were predicted through small RNA sequencing, many exclusively identified within the presence or absence of bromide. Together, the miRNA analyses across this chapter and the previous chapter highlighted the potential for species-specific post-transcriptional regulation in A. taxiformis. Overall, this thesis explored the complex molecular and environmental controls regulating halogenated compound production in A. taxiformis. This growing body of research not only enhances our understanding of red seaweed biology but also informs aquaculture strategies aimed at optimising A. taxiformis cultivation for sustainable methane mitigation in agriculture. The experimental data produced by this thesis contributes to the development of A. taxiformis as a molecular model for red seaweed biology, with particular consideration to the novel investigation into its miRNA landscape. The outcomes of this thesis provide significant foundational research through A. taxiformis transcriptomics and miRNA profiling, while delivering key molecular insights into halogenation within the seaweed.

Molecular analysis of bromoform biosynthesis in the red seaweed Asparagopsis taxiformis

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