Identification of Delta12-fatty acid desaturase from arachidonic acid-producing mortierella fungus by heterologous expression in the yeast Saccharomyces cerevisiae and the fungus Aspergillus oryzae.
Based on the sequence information for the omega3-desaturase genes (from Brassica napus and Caenorhabditis elegans), which are involved in the desaturation of linoleic acid (Delta9, Delta12-18 : 2) to alpha-linolenic acid (Delta9, Delta12, Delta15-18 : 3), a cDNA was cloned from the filamentous fungal strain, Mortierella alpina 1S-4, which is used industrially to produce arachidonic acid. Homology analysis with protein databases revealed that the amino acid sequence showed 43.7% identity as the highest match with the microsomal omega6-desaturase (from Glycine max, soybean), whereas it exhibited 38.9% identity with the microsomal omega3-desaturase (from soybean). The evolutionary implications of these enzymes will be discussed. The cloned cDNA was confirmed to encode a Delta12-desaturase, which was involved in the desaturation of oleic acid (Delta9-18 : 1) to linoleic acid, by its expression in both the yeast Saccharomyces cerevisiae and the fungus Aspergillus oryzae. Analysis of the fatty acid composition of yeast and fungus transformants demonstrated that linoleic acid (which was not contained in the control strain of S. cerevisiae) was accumulated in the yeast transformant and that the fungal transformant contained a large amount of linoleic acid (71.9%). Genomic Southern blot analysis of the transformants with the Mortierella Delta12-desaturase gene as a probe confirmed integration of this gene into the genome of A. oryzae. The M. alpina 1S-4 Delta12-desaturase is the first example of a cloned nonplant Delta12-desaturase.
Sakuradani
E
E
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Osaka, Japan.
Kobayashi
M
M
Ashikari
T
T
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; D017931:DNA Primers; D018076:DNA, Complementary; D044943:Fatty Acid Desaturases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D020094:Mortierella / Q000201:enzymology*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D016718:Arachidonic Acid; D044943:Fatty Acid Desaturases; C040448:delta-12 fatty acid desaturase