Delta6-fatty acid desaturase from an arachidonic acid-producing Mortierella fungus. Gene cloning and its heterologous expression in a fungus, Aspergillus.
A DNA fragment was cloned from the fungal strain, Mortierella alpina 1S-4 (which is used industrially to produce arachidonic acid), after PCR amplification with oligonucleotide primers designed based on the sequence information for delta6-desaturase genes (from borage and Caenorhabditis elegans), which are involved in the desaturation of linoleic acid (delta9, delta12-18:2) to gamma-linolenic acid (delta6, delta9, delta12-18:3). This fragment was used as a probe to isolate a cDNA clone with an open reading frame encoding 457 amino acids from a M. calpina 1S-4 library. The predicted amino-acid sequence showed similarity to those of the above delta6-desaturases, and contained a cytochrome b5-like domain at the N-terminus, being different from the yeast delta9-desaturase which has the corresponding domain at the C-terminus. The full-length cDNA clone was expressed under the control of the amyB promoter in a filamentous fungus, Aspergillus oryzae, resulting in the accumulation of gamma-linolenic acid (which was not detected in the control Aspergillus) to the level of 25.2% of the total fatty acids. These findings revealed that the recombinant product has delta6-desaturase activity. The Mortierella delta6-desaturase is the first to be reported in fungi.
Sakuradani
E
E
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan.
Kobayashi
M
M
Shimizu
S
S
D000595:Amino Acid Sequence; D016718:Arachidonic Acid / Q000096:biosynthesis*; D001236:Aspergillus oryzae / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018076:DNA, Complementary; D044943:Fatty Acid Desaturases / Q000235:genetics / Q000302:isolation & purification*; D008401:Gas Chromatography-Mass Spectrometry; D050602:Linoleoyl-CoA Desaturase; D008055:Lipids / Q000032:analysis; D008969:Molecular Sequence Data; D020094:Mortierella / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D016366:Open Reading Frames; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D008055:Lipids; D016718:Arachidonic Acid; D044943:Fatty Acid Desaturases; D050602:Linoleoyl-CoA Desaturase