title: Cloning and functional identification of delta5 fatty acid desaturase gene and its 5'-upstream region from marine fungus Thraustochytrium sp. FJN-10.
abstract: 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.
authors.affiliation: Engineering Research Center of Industrial Microbiology, Ministry of Education, Fujian Normal University, Fuzhou, Fujian, 350108, People's Republic of China. hjz@fjnu.edu.cn
authors.forename: Jian-Zhong
authors.initials: JZ
authors.lastname: Huang
authors.forename: Xian-Zhang
authors.initials: XZ
authors.lastname: Jiang
authors.forename: Xiao-Feng
authors.initials: XF
authors.lastname: Xia
authors.forename: Ai-Qun
authors.initials: AQ
authors.lastname: Yu
authors.forename: Ruo-Yu
authors.initials: RY
authors.lastname: Mao
authors.forename: Xiao-Feng
authors.initials: XF
authors.lastname: Chen
authors.forename: Bao-Yu
authors.initials: BY
authors.lastname: Tian
mesh_terms: 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
chemical_list: D005231:Fatty Acids, Unsaturated; D005656:Fungal Proteins; D044943:Fatty Acid Desaturases