Cloning and functional identification of delta5 fatty acid desaturase gene and its 5'-upstream region from marine fungus Thraustochytrium sp. FJN-10.
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-γ-linolenic acid (20:3 Δ(8,11,14)), resulting in arachidonic acid (20:4 Δ(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.
Huang
Jian-Zhong
JZ
Engineering Research Center of Industrial Microbiology, Ministry of Education, Fujian Normal University, Fuzhou, Fujian, 350108, People's Republic of China. hjz@fjnu.edu.cn
Jiang
Xian-Zhang
XZ
Xia
Xiao-Feng
XF
Yu
Ai-Qun
AQ
Mao
Ruo-Yu
RY
Chen
Xiao-Feng
XF
Tian
Bao-Yu
BY
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D044943:Fatty Acid Desaturases / Q000235:genetics* / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005658:Fungi / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016364:Reading Frames; D058009:Stramenopiles / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification
D005231:Fatty Acids, Unsaturated; D005656:Fungal Proteins; D044943:Fatty Acid Desaturases