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Production of poly-unsaturated fatty acids by Glossomastix chrystoplastos - by Tracy Hsiao

Characterization of microalga Glossomastix chrysoplastos, an eicosapentaenoic acid (EPA) producer in class, Pinguiophyceae, of the division Chromophyte, was performed to maximize algal growth and its EPA production. In addition to fermentation optimization, desaturase gene in G. chrysoplastos will also be identified to study the relationship between EPA production and desaturase expression in different fermentation conditions.

Algae in the class Pinguiophyceae characteristically produce large amounts of EPA, and they are phylogeneticly distinct from the algae species in the other classes. EPA is an important omega-3 polyunsaturated fatty acid containing twenty carbons and five double bonds in cis formation. According to the American Heart Association1, large-scale epidemiological studies suggest that people at risk for coronary heart disease benefit from consuming omega-3 polyunsaturated fatty acids from plants and marine sources. Only selected organisms such as algae and fungus can produce long chain polyunsaturated fatty acids such as eicosapentaenoic acid in a de novo process, as all other organisms lack all the required position specific desaturases (Figure 1). Human can produce EPA by ingesting plants with the correct unsaturated fatty acids, but the conversion rate is modest and controversial. Although synthesis of fatty acids containing less than eighteen-carbons is well understood in higher plants and algae, the knowledge of the long chain polyunsaturated fatty acid in algae is still under investigation.

The effect of environmental factors such as dilution rate, pH, and temperature on the algae lipid production, especially EPA, was investigated in a continuous photobioreactor. Lipid fractionation demonstrated that G. chrysoplastos contains mostly glycolipid, and most EPA present in the glycolipid (monogalactosyldiacylglycerol lipid) fraction. The observation coincides with our hypothesis that EPA functions as structure lipids in algae.

In additional to growth parameter studies, intermediates fed studies indicated that G. chrysoplastos utilize the traditional fatty acid synthase pathway for fatty acid synthesis. In order to study EPA production in the metabolic level, D5 and D6 desaturases ( Figure 2) in G. chrysoplastos are in the process of being isolated from its mRNA through RT-PCR (reverse transcription- polymerase chain reaction). The probes for fatty acid desaturase are generated by reverse transcription of algal RNA with poly-A primers and PCR amplification with degenerate primers designed from conserved histidine regions of desaturases in other species. A cDNA library constructed with the algal mRNA is used to identify the full-length gene by the fatty acid desaturase probe, and gene transformation into yeast provides functional analysis for further identification.

Figure 1: Position specific desaturases for fatty acids desaturation.





Figure 2.  Possible EPA synthesis pathways

E stands for enlongase, D5, D6 and w3 stand for position specific desaturases. Fatty acids is denoted as (A:BwC): A stands for the number of carbons, B stands for number of double bonds, and C stands for the position of first double bond from the methyl ends.

Reference:Circulation, 106:2747-2757, 2002.



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