期刊
MARINE BIOTECHNOLOGY
卷 13, 期 1, 页码 12-21出版社
SPRINGER
DOI: 10.1007/s10126-010-9262-6
关键词
Desaturase; Promoter; Thraustochytrium; Polyunsaturated fatty acid; Docosahexaenoic acid
资金
- National Natural Science Foundation of China [30370028, 30970047]
- Natural Science Foundation of Fujian, China [2003F005, 2008F3036]
- Fujian Development and Reform Commission [[2005] 847, 2005Q007]
A gene encoding delta5 fatty acid desaturase (fad5) was cloned from marine fungus Thraustochytrium sp. FJN-10, a species capable of producing docosahexaenoic acid. The open reading frame of fad5 was 1,320 bp and encoded a protein comprising 439 amino acids. Expression of the fad5 in Saccharomyces cerevisiae INVSC1 revealed that FAD5 is able to introduce a double bond at position 5 of the dihomo-gamma-linolenic acid (20:3 Delta(8,11,14)), resulting in arachidonic acid (20:4 Delta(5,8,11,14)) with a conversion rate of 56.40% which is the highest among engineering yeasts reported so far. The 5'-upstream region of fad5 was cloned by LA-PCR and analyzed. Phylogenetic analysis of this sequence with the 5'-upstream region of other delta5 desaturases showed that the 5'-upstream region of fad5 from Thraustochytrium share the smallest evolution distance with human and rhesus. Computational analysis of the nucleotide sequence of the 5'-upstream region of fad5 has revealed several basic transcriptional elements including five TATA boxes, three CCAAT boxes, 12 GC boxes, and several putative target-binding sites for transcription factors such as HSF, CAP, and ADR1. Preliminary functional analysis of this promoter in S. cerevisiae shows that the 5'-upstream region of fad5 could drive the expression of green fluorescent protein.
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