title: Isolation and characterization of the diatom Phaeodactylum Δ5-elongase gene for transgenic LC-PUFA production in Pichia pastoris.
abstract: The diatom Phaeodactylum tricornutum can accumulate eicosapentaenoic acid (EPA) up to 30% of the total fatty acids. This species has been targeted for isolating gene encoding desaturases and elongases for long-chain polyunsaturated fatty acid (LC-PUFA) metabolic engineering. Here we first report the cloning and characterization of Δ5-elongase gene in P. tricornutum. A full-length cDNA sequence, designated PhtELO5, was shown to contain a 1110 bp open reading frame encoding a 369 amino acid polypeptide. The putative protein contains seven transmembrane regions and two elongase characteristic motifs of FLHXYHH and MYSYY, the latter being typical for microalgal Δ5-elongases. Phylogenetic analysis indicated that PhtELO5 belongs to the ELO5 group, tightly clustered with the counterpart of Thalassiosira pseudonana. Heterologous expression of PhtELO5 in Pichia pastoris confirmed that it encodes a specific Δ5-elongase capable of elongating arachidonic acid and eicosapentaenoic acid. Co-expression of PhtELO5 and IsFAD4 (a ∆4-desaturase from Isochrysis sphaerica) demonstrated that the high-efficiency biosynthetic pathway of docosahexaenoic acid was assembled in the transgenic yeast. Substrate competition revealed that PhtELO5 exhibited higher activity towards n-3 PUFA than n-6 PUFA. It is hypothesized that Phaeodactylum ELO5 may preferentially participate in biosynthesis of transgenic LC-PUFA via a n-3 pathway in the yeast host.
authors.affiliation: Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, Wuhan 430062, China. mljiang@oilcrops.cn.
authors.forename: Mulan
authors.initials: M
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, Wuhan 430062, China. tcguobing@126.com.
authors.forename: Bing
authors.initials: B
authors.lastname: Guo
authors.affiliation: Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, Wuhan 430062, China. wanxia@oilcrops.cn.
authors.forename: Xia
authors.initials: X
authors.lastname: Wan
authors.affiliation: Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, Wuhan 430062, China. gongyangmin@caas.cn.
authors.forename: Yangmin
authors.initials: Y
authors.lastname: Gong
authors.affiliation: Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, Wuhan 430062, China. zhangyinbo@caas.cn.
authors.forename: Yinbo
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, Wuhan 430062, China. huchuanjiong@caas.cn.
authors.forename: Chuanjiong
authors.initials: C
authors.lastname: Hu
mesh_terms: D000123:Acetyltransferases / Q000235:genetics*; D000595:Amino Acid Sequence; D003470:Culture Media; D017931:DNA Primers; D017377:Diatoms / Q000235:genetics*; D004281:Docosahexaenoic Acids / Q000378:metabolism; D019143:Evolution, Molecular; D044943:Fatty Acid Desaturases / Q000378:metabolism; D000081031:Fatty Acid Elongases; D005227:Fatty Acids / Q000032:analysis / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000096:biosynthesis*; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D030781:Organisms, Genetically Modified; D010802:Phylogeny; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D014170:Transformation, Genetic
chemical_list: D003470:Culture Media; D017931:DNA Primers; D005227:Fatty Acids; D005231:Fatty Acids, Unsaturated; D007202:Indicators and Reagents; D004281:Docosahexaenoic Acids; D044943:Fatty Acid Desaturases; C434953:delta-4 fatty acid desaturase; D000123:Acetyltransferases; D000081031:Fatty Acid Elongases