Expression of fungal desaturase genes in cultured mammalian cells.
Long-chain polyunsaturated fatty acids (LC-PUFA) are important components of cellular structure and function. Most of LC-PUFA are derived from linoleic acid and alpha-linolenic acid. In plants and fungi, these two acids can be synthesized from oleic acid via the action of two enzymes, delta12 and delta15-desaturases. Due to lack of these enzymatic activities and the ability to synthesize these two essential fatty acids, animals must obtain them from the diet. In this report, we demonstrated the expression of a fungal delta12-desaturase gene in mouse L cells incubated in serum-free medium. The results showed a significant increase in the amount of linoleic acid with a concomitant decrease of oleic acid in cellular lipids. Most of the newly formed linoleic acid was incorporated into cellular phospholipids, particularly phosphatidylcholine. The increase of linoleic acid provided the substrate for the endogenous synthesis of (n-6) LC-PUFA, such as eicosadienoic acid (EDA), dihomo-gamma-linoleic acid (DGLA) and arachidonic acid (AA). Prolonged incubation further increased the levels of linoleic acid derived from oleic acid by the action of delta12-desaturase, and the levels of 20:2n-6 produced from linoleic acid by the action of the endogenous elongase. However, prolonged incubation suppressed significantly the formation of DGLA and AA. In a separate study, a fungal delta6-desaturase gene has also been expressed in the mouse L cells incubated in serum-containing medium. The result shows a significant increase in levels of 20:3n-6 and 20:4n-6. These findings demonstrate that through genetic modification, it is possible to (1) generate cell lines which no longer require dietary 'essential' fatty acids and (2) alter the endogenous fatty acid metabolism to enhance the production of LC-PUFA and their derivatives.
Kelder
B
B
Edison Biotechnology Institute and Department of Biomedical Sciences, Ohio University, Athens, USA.
Mukeji
P
P
Kirchner
S
S
Hovanec
G
G
Leonard
A E
AE
Chuang
L T
LT
Kopchick
J J
JJ
Huang
Y S
YS
D000818:Animals; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000235:genetics; D044943:Fatty Acid Desaturases / Q000235:genetics / Q000378:metabolism*; D005228:Fatty Acids, Essential / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000096:biosynthesis* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005800:Genes, Fungal / Q000502:physiology*; D006801:Humans; D007739:L Cells; D019787:Linoleic Acid / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D020094:Mortierella / Q000235:genetics*; D019301:Oleic Acid / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D013997:Time Factors; D014162:Transfection; D014170:Transformation, Genetic; D017962:alpha-Linolenic Acid / Q000378:metabolism
D005228:Fatty Acids, Essential; D005231:Fatty Acids, Unsaturated; D017962:alpha-Linolenic Acid; D019301:Oleic Acid; D019787:Linoleic Acid; D044943:Fatty Acid Desaturases