Molecular analysis of front-end desaturases in the biosynthesis of polyunsaturated fatty acids in microalga Prorocentrum micans
Date
2024-04-15
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ORCID
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Thesis
Degree Level
Masters
Abstract
The marine dinoflagellate Prorocentrum micans produces a high amount of 3 polyunsaturated fatty acids (PUFAs), including stearidonic acid (SDA, 18:4n-3), octadecapentaenoic acid (OPA, 18:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). However, the mechanisms of how these PUFAs are synthesized in P. micans remain unknown. Sequence analysis of transcriptomic data of P. micans initially indicated that the aerobic pathway might be responsible for the biosynthesis of PUFAs. The study was thus subsequently focused on the front-end desaturases in the aerobic pathway introducing carbon-carbon double bonds between the carboxyl end and the pre-existing double bond in a fatty acid chain. Five candidate genes were cloned out from complementary DNA (cDNA) by reverse transcription polymerase chain reaction (RT-PCR), and functions of these genes were then characterized by heterologous expression in yeast. One candidate gene, designated PmD6, was found to encode a functional 6 fatty acid desaturase with a strong substrate preference toward alpha-linolenic acid (ALA, 18:3n-3) over linoleic acid (LA, 18:2n-6). To further investigate the structure-function relationship of PmD6, structure alignment between the crystal structure of stearoyl-CoA desaturase and modelled structure of PmD6 predicted by AlphaFold was performed. Critical residues involved in forming the substrate tunnel and stabilizing the regional structure of the catalytic site in PmD6 were determined, and their functional roles were analyzed by yeast expression of the mutants generated by site-directed mutagenesis. Residues Tyr226, Trp235 and Phe345 were found to be involved in defining the substrate tunnel of the middle region, while Leu391 and Met389 were shown to engage in forming the methyl-end region. Asp255, positioning near the catalytic centre, was found to be absolutely required in stabilizing the regional structure for catalytic activity.
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Keywords
Prorocentrum micans, front-end desaturases, polyunsaturated fatty acids
Citation
Degree
Master of Science (M.Sc.)
Department
Food and Bioproduct Sciences
Program
Food Science