Functional characterization of polyunsaturated fatty acid delta 6-desaturase and elongase genes from the black seabream (Acanthopagrus schlegelii).
Fatty acid delta 6-desaturase (D6DES) and elongases are key enzymes in the synthesis of polyunsaturated fatty acids (PUFAs) including arachidonic acid (ARA) and eicosapentaenoic acid (EPA) from microorganisms to higher animals. To identify the genes encoding D6DES and elongases for PUFAs, we isolated each cDNA with a high similarity to the D6DES and ELOVL5-like elongases of mammals and fishes via degenerate PCR and RACE-PCR from Acanthopagrus schlegelii. A recombinant vector expressing AsD6DES was subsequently constructed and transformed into Saccharomyces cerevisiae to test the enzymatic activity toward n-6 and n-3 fatty acids in the PUFA biosynthesis. The heterologously expressed AsD6DES produced γ-linolenic acid (GLA, C18:3 n-6) and stearidonic acid (STA, C18:4 n-3) at conversion rates of 26.3-35.6% from exogenous linoleic acid (LA, C18:2 n-6) and α-linolenic acid (ALA, C18:3 n-3) substrates, respectively. When AsELOVL5 was expressed in yeast, it conferred an ability to elongate GLA to di-homo-γ-linolenic acid (DGLA, C20:3 n-6). In addition, AsELOVL5 showed an ability to convert ARA (C20:4 n-6) and EPA (C20:5 n-3) to dodecylthioacetic acid (DTA, C22:4 n-6) and docosapentaenoic acid (DPA, C22:5 n-3), respectively. In these results, the AsD6DES encodes a delta 6-fatty acid desaturase and the AsELOVL5 encoding a long-chain fatty acid elongase shows activity to enlongate C18Δ6/C20Δ5, but not C22.
Rural Development Administration, National Academy of Agricultural Science, Seodun-Dong, Suwon, 441-707, Korea, sunheekim00@korea.kr.
Sun Hee
SH
Kim
Jong Sug
JS
Park
So Yun
SY
Kim
Jung-Bong
JB
Kim
Kyung Hee
KH
Roh
Hyun Uk
HU
Kim
Kyeong-Ryeol
KR
Lee
Jong Bum
JB
Kim
D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000096:biosynthesis; D002849:Chromatography, Gas; D003001:Cloning, Molecular; D015118:Eicosapentaenoic Acid / Q000096:biosynthesis; D000081031:Fatty Acid Elongases; D005227:Fatty Acids / Q000032:analysis; D015525:Fatty Acids, Omega-3 / Q000096:biosynthesis; D050602:Linoleoyl-CoA Desaturase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D021541:Sea Bream / Q000145:classification / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity; D017965:gamma-Linolenic Acid / Q000096:biosynthesis
D005227:Fatty Acids; D015525:Fatty Acids, Omega-3; D016718:Arachidonic Acid; D017965:gamma-Linolenic Acid; D015118:Eicosapentaenoic Acid; D050602:Linoleoyl-CoA Desaturase; D000123:Acetyltransferases; D000081031:Fatty Acid Elongases; C062895:stearidonic acid