Molecular Mechanism Of DHA Biosynthesis In Marine Fungi Thraustochytrids

Docosahexaenoic acid (DHA C22:6n-3), a typical long chain polyunsaturated fatty acids (PUFAs) has attracted much attention due to its many positive effects on diseases such as artherosclerosis, hypertriglyceridemia, hypertension and cancers. DHA is not synthesized in adequate amounts to meet metabolic demands of the human body thus must be obtained from dietary sources. The traditional sources of DHA are fish oils. However conventional fish oils may not be suitable to meet the increasing market for DHA owing to their poor quality and low quantity. Marine fungi especially Thraustochytrids producing a significant amount of DHA can serve as model organisms for explaining the mechanism on the biosynthesis of PUFA. In this paper, the following findings were ascribed:1. Two elongase genes (TFD6 and TFD5) involved in the biosynthesis of DHA in Thraustochytrium sp. FJN-10 were cloned by using reverse transcription PCR and rapid amplification of cDNA ends. TFD6 cDNA was 816 bp in length and encoded a protein of 271 amino acids. TFD5 cDNA was 831 bp in length and encoded a protein of 276 amino acids. Transmembrane analysis revealed that TFD6 contained five transmembrane domains while TFD5 contained seven. Tertiary structures of TFD6, TFD5 elongases were predicted by HHMMSTR (Hidden markov model for local sequence-structure) model and Rosetta program. Alignment of TFD6, TFD5 with other elongases showed that both of them shared a HXXHH conserved histidine-rich motif. Phylogenetic analysis showed TFD6 was the closest to Thraustochytrium A 6 elongase, while TFD5 was the closest to Thraustochytrium sp. A 5 elongase. TFD6 and TFD5 were subcloned into the Hind III /Xba I restriction site of pYES2 vector respectively. Recombined plasmids were transformed into Saccharomyces cerevisiae using lithium acetate method. Heterologous expression systems were verified by PCR and sequence analysis. Gas chromatogrms showed TFD6 could elongate C18:3n-3 to C20:3n-3 while TFD5 could elongate C20:5n-3 to C22:5n-3.2. Marine fungi Schizochytrium sp. FJU-512 accumulating significant amounts of DHA was isolated by using pine pollen as bait. Its single cell oil was very valuable since...