[Isolation and functional identification of delta 5 desaturase gene from Mortierella alpina].
Arachidonic acid is an essential fatty acid in human nutrition and a biogenetic precursor of the biologically active prostaglandins and leukotrienes. delta5 desaturase is a key enzyme in the biosynthetic pathway of arachidonic acid, which catalyze the delta5 dehydrogenation of di-homo-gamma-linolenic acid to form arachidonic acid. Complete cDNA encoding putative delta5 fatty acyl desaturase was isolated from Mortierella alpina M6 via RT-PCR. The full length cDNA consisted of 1366 nucleotides encoding 446 amino acids. The deduced protein had conserved domains of known delta5 fatty acyl desaturases including a cytochrome b5 domain in the N terminal and 3 conserved histidine box. To elucidate the function of the protein, the cDNA was subcloned into the expression vector pPIC9K. The resultant recombinant plasmid pPIC9K-D5 was transformed to Pichia pastoris GS115 by electroporation. Transformants containing multi-copy delta5 desaturase gene were screened by Geneticin resistance. The transformants were induced to express the inserted gene with methanol when di-homo-gamma-linolenic acid was provided as an exogenous substrate. Analysis of the recombinant yeast lipids by gas chromatograph showed that a novel peak corresponding to the standard of arachidonic acid was detected. The novel peak was further characterized by GC-MS and identified as arachidonic acid. The results showed that the gene isolated from fungi Mortierella alpina M6 was a delta5 desaturase gene.
Zhu
Min
M
Huazhong University of Science and Technology, School of Life Science and Technology, Wuhan 430074, China. zhumin@mail.nust.edu.cn
Liu
Zhi
Z
Yu
Long-Jiang
LJ
Zhu
Lu
L
Cheng
Hua
H
D000595:Amino Acid Sequence; D016718:Arachidonic Acid / Q000032:analysis / Q000096:biosynthesis; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D000090943:Delta-5 Fatty Acid Desaturase; D044943:Fatty Acid Desaturases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D008969:Molecular Sequence Data; D020094:Mortierella / Q000201:enzymology / Q000235:genetics*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic
D018076:DNA, Complementary; D000090943:Delta-5 Fatty Acid Desaturase; D005656:Fungal Proteins; D011994:Recombinant Proteins; D016718:Arachidonic Acid; D044943:Fatty Acid Desaturases