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Phytosterol production from Phaeodactylum tricornutum: Insights into environmental conditions and their effect on gene expression

Randhir, Ankitha (2020) Phytosterol production from Phaeodactylum tricornutum: Insights into environmental conditions and their effect on gene expression. PhD thesis, Murdoch University.

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Abstract

Phytosterols are known to have cholesterol-lowering, anti-inflammatory, anti-cancer, anti-atherogenic, neuro modulatory and neuro-protective activities which has led to their use in the pharmaceutical, neutraceutical and functional food industries. Higher plants are the current commercial source of phytosterols. Production of the oil obtained from higher plants may not be able to keep up with probable increases in phytosterol demand. Additionally, the extensive need for agricultural land and freshwater to grow higher plants to obtain phytosterols is not sustainable and hence there is a need for an alternate source. Microalgae produce phytosterols that are reported to have the same efficacy as those isolated from higher plants making this a potential alternative source of these compounds. Previous studies have noted that phytosterol content and composition in microalgae changes based on light, nutrient concentrations, salinity and temperature. However, only a few studies have been carried out on microalgae focusing on the effects of these culture conditions on the phytosterol content. This information is essential to assess the potential of microalgae as an alternate commercial source of these important compounds.

Phaeodactylum tricornutum is known to produce phytosterols of commercial interest such as brassicasterol and campesterol. In addition, the availability of whole-genome sequence data, a putative sterol biosynthetic pathway and well-established molecular techniques provides a unique opportunity to map changes in P. tricornutum culture to changes in gene expression. This makes P. tricornutum a suitable candidate for phytosterol production studies.

At present, the sterol biosynthetic pathway in P. tricornutum has been proposed based on in silico analysis and is not fully validated. The proposed biosynthetic pathway indicates that enzymes delta 7-reductase and C22-desaturase are involved in the conversion of ergosterol to campesterol and campesterol to brassicasterol respectively. A methodology for determining the expression profile of the two genes coding for the enzymes delta 7-reductase and C22-desaturase was developed and used for monitoring. To the best of my knowledge, this is the first time that delta 7-reductase gene expression in P. tricornutum has been studied.

To understand the influence of various culture parameters such as light, nutrient concentrations, salinity and temperature for improved phytosterol content in P. tricornutum, the culture was grown under a) white light and monochromic red light at 60, 160 and 260 μmol photons.m-2.s-1, b) nitrogen and phosphorus concentrations 2 (F) and 4 (2F) times higher than modified F/2 culture media, c) salinity levels of 25 and 35 g.L-1 NaCl, and d) culture temperatures of 15 and 25 °C. The relative gene expression of delta 7-reductase and C22-desaturase was also studied for the various nutrient and salinity conditions.

The results indicated that the light irradiance (60, 160, and 260 μmol photons.m-2.s-1), nitrogen and phosphorus concentrations (F/2, F and 2F) and temperature (15 and 25 ± 3 °C) individually do not have any significant influence on the phytosterol content of P. tricornutum CS-29/7. On the other hand, phytosterol content of P. tricornutum CS-29/7 when grown using white light at 60 and 260 μmol photons.m-2.s-1 resulted in 3 times higher phytosterol content than the cells grown under similar monochromatic red lights. Salinity levels (25 and 35 g.L-1 NaCl) influence the phytosterol content of P. tricornutum with 52 % less phytosterol content at the stationary phase of growth in 35 g.L-1 than 25 g.L-1 salinity. Also, an interactive effect of salinity (25 g.L-1 NaCl) and temperature (15 ± 3 °C) was observed that enhanced the phytosterol content 1.75 times compared to the cells grown at 35 g.L-1 NaCl at 15 ± 3 °C. Growth phase can also influence the phytosterol content in P. tricornutum CS-29/7. Under red light conditions (60 and 260 μmol photons.m-2.s-1), 3 - 4 times higher phytosterol content (mg.g-1 AFDW) was observed when the culture progressed from log to stationary phase. On the other hand, when grown at 35 g.L-1 NaCl salinity, phytosterol content was observed to decrease by half when the culture progressed from log to stationary phase. Brassicasterol was the only sterol identified in all the studies except salinity studies where the presence of ergosterol was also detected in both 25 and 35 g.L-1 NaCl conditions.

The expression of the C22 desaturase gene was downregulated when the nutrient concentrations were increased whereas, the delta 7-reductase gene showed varied expression at the log and late log phases of growth. Under the various salinity conditions, both the genes were upregulated when the salinity level was reduced to 25 g.L-1 NaCl. The results of the microalgal culture optimisation experiments and the enzyme expression were then combined. Surprisingly, the expression of delta 7- reductase and C22-desaturase was not correlated to changes in phytosterol synthesis. This suggests that the proposed biosynthetic pathway in P. tricornutum may need to be revised.

Considering all the culture parameters tested, it was found that salinity and temperature together were the most important factors for improving phytosterol accumulation. Culturing the algae under optimised conditions of salinity (25 g.L-1 NaCl) and temperature (15 °C) in modified 2F media at 300 μmolphotons.m-2.s-1 resulted in phytosterol content and maximum phytosterol productivity of 2.8 mg.g-1 AFDW and 340 μg.L-1.day-1 respectively.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): Environmental and Conservation Sciences
Supervisor(s): Moheimani, Navid and Laird, Damian
URI: http://researchrepository.murdoch.edu.au/id/eprint/60932
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