Genome Guided Enzyme Discovery in the Extremophile Galdieria sulphuraria

Galdieria sulphuraria is a eukaryotic unicellular red alga that predominates geothermal
sites with low pH (0-2) and high temperatures (50-56 °C), the absolute limits of eukaryotic
life. It can grow photoautotrophically and use a vast array of sugars, organic acids and
polyols to support both heterotrophic and mixotrophic growth; making this alga an
interesting focus for investigation on novel enzyme discovery. Based on its lifestyle, it
seems likely that its enzymes may to be highly thermostable (for a eukaryote) and its
secreted enzymes should display high acid tolerance. Consequently, any protein
products discovered are likely to be robust and well suited for industrial biotechnology
(IB) applications.

Firstly, I resolved the nuclear phylogeny and investigated evolutionary pressures acting
on the Galdieria genus. This revealed the subdivision of the G. sulphuraria into six
lineages. Analysis of dN/dS rates showed different evolutionary pressures acting
between the strains and revealed a selection of genes under positive selection, one of
these had predicted involvement in the degradation of lignocellulosic material. Secondly,
I obtained transcriptomic and long read DNA sequence data to annotate the core six
genomes, subsequent extensive CAZymes analysis showed ~128 enzymes per strain.
Fewer than expected CAZyme families were represented given G. sulphuraria’s
extraordinary growth capacity. This led to investigating heterotrophic growth on different
carbohydrate polymers to identify industrially relevant secreted enzymes. Extraction of
proteins from the supernatants and analysis via LCMS showed the presence of
potentially interesting enzymes, prompting further investigation. Three target genes were
identified and selected for heterologous expression and characterisation. A previously
uncharacterised gene was successfully purified and refolded at pH 2 and shown to
denature at 92 °C. There is scope to develop G. sulphuraria’s acid tolerant, thermostable
proteins for industrial use. This thesis has expanded understanding of this extremophile
and identified multiple novel enzymes with potential for industrial development.