LE3 .A278 2017

2017

Merkx-Jacques, Alexandra
Lopez, Juan Carlos

Acadia University

Bachelor of Science

Honours

Biology

Biology

Single celled eukaryotes such as Thraustochytrid T18 may be a more economic and environmentally friendly source of omega-3 fatty acids. To decrease fermentation costs, genes involved in the digestion of hemicellulosic waste derived carbohydrates has been added to T18, to produce several strains, including 7-7 and 51-7. Observations of 51-7 and 7-7 have previously shown to have reduced growth during the later stages of fermentation presumably related to nitrogen depletion. As the α-tubulin promoter controls the expression of the transgenes, reduced promoter activity in the low nitrogen conditions present late in the fermentation could explain the reduced growth. To measure α-tubulin expression, xylose depletion assays were run on strains with two nitrogen concentration groups, with the understanding that if α-tubulin was downregulated the xylose isomerase (XI) and xylulose kinase (XK) transgene expression would be decreased resulting in reduced xylose consumption, enzyme activity of XI and XK was also measured via whole cell extract. Additionally, strains encoding luciferase genes were grown in different nitrogen conditions and the luciferase expression was measured. Contrary to expectation, the consumption of xylose in the low nitrogen media initially increased, as did the expression of the α-tubulin controlled transgenes, there was a build up of xylitol however. The low nitrogen conditions appeared to cause the oxidoreductive xylose metabolism pathway to be unable to dehydrate xylitol into xylulose, arresting the culture’s growth, providing an explanation for 7-7 and 51-7’s reduced growth during the late stages of fermentations.

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https://scholar.acadiau.ca/islandora/object/theses:2709