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Isolation and functional characterization of polyunsaturated fatty acid elongase (AsELOVL5) gene from black seabream (Acanthopagrus schlegelii)

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Abstract

To identify the genes encoding fatty acid elongases for the biosynthesis of polyunsaturated fatty acids (PUFAs), we isolated a cDNA via degenerate PCR and RACE-PCR from Acanthopagrus schlegelii with a high similarity to the ELOVL5-like elongases of mammals and fishes. This gene is termed AsELOVL5 and encodes a 294 amino acid protein. When AsELOVL5 was expressed in Saccharomyces cerevisiae, it conferred an ability to elongate γ-linolenic acid (18:3 n−6) to di-homo-γ-linolenic acid (20:3 n−6). In addition, the transformed cells converted arachidonic acid (20:4 n−6) and eicosapentaenpic acid (20:5 n−3) to docosatetraenoic acid (22:4 n−6) and docosapentaenoic acid (22:5 n−3), respectively. These results indicate that the AsELOVL5 gene encodes a long-chain fatty acid elongase capable of elongating C18Δ6/C20Δ5 but not C22 PUFA substrates.

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Acknowledgments

This work was supported by grants from the National Academy of Agricultural Science (PJ006745), the Rural Development Administration, Republic of Korea. S.H. Kim was supported by a 2011 Post-doctoral Fellowship Program of National Academy of Agricultural Science, the Rural Development Administration, Republic of Korea.

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Authors and Affiliations

  1. National Academy of Agricultural Science, Rural Development Administration, Seodun-Dong, Suwon, 441-707, Korea

    Sun Hee Kim, Jong Bum Kim, So Yun Kim, Kyung Hee Roh, Hyun Uk Kim, Kyeong-Ryeol Lee & Jong-Sug Park

  2. East Sea Environment Research Department, KORDI, 695-1, Hujung-ri, Jukbyeon-myun, Uljin-gun, Gyeongbuk, Seoul, 767-813, Korea

    Yo Soon Jang

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  1. Sun Hee Kim
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Corresponding author

Correspondence to Jong-Sug Park.

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Sun Hee Kim and Jong Bum Kim contributed equally to this work.

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Fig. S1

Alignment of the deduced amino acid sequence of Acanthopagrus schlegelii polyunsaturated fatty acid elongase-like cDNA with those of human and fishes. The putative histidine box motif is lined with dot. Putative membrane-spanning domains are marked from I to V with underlines. The leftwards and rightwards arrows indicate the binding locations of the two degenerate primers. Sequence identities between Acanthopagrus schlegelii and the other species are black boxed with white letters. AsELOVL5 (Black seabream, Acanthopagrus schlegelii, Bankit gb|1218654|), SaELO (Gilthead seabream, Sparus aurata fatty acid elongase, AAT81404), TmELOVL5 (Southern Bluefin Tuna, Thunnus maccoyii fatty acid elongase, ACZ55930), SsElovl5b (Alantic Salmon, Salmo salar polyunsaturated fatty acid elongase, NP_001130024), SsElovl5a (Alantic Salmon, Salmo salar polyunsaturated fatty acid elongase, NP_001117039), OmELO (Rainbow trout, Oncorhynchus mykiss polyunsaturated fatty acid elongase, NP¬_001118108), DrELOVL5 (Zebrafish, Danio rerio polyunsaturated fatty acid elongase, NP_956747), HsELOVL5 (Human, Homo sapiens very long chain fatty acids elongase, NP_068586), SsELOVL2 (Alantic Salmon, Salmo salar polyunsaturated fatty acid elongase, NP_001130025), and HsELOVL2 (Human, Homo sapiens very long chain fatty acids elongase, NP_060240) (TIFF 1769 kb)

Fig. S2

Phylogenetic tree of fatty acid elongases from A. schlegelii (AsELOVL5), other fish species and human. Sequence alignments and phylogenetic tree construction were performed using CLUSTAL W. The horizontal branch length is proportional to the amino acid substitution rate per site. The numbers represent the frequencies with which the tree topology presented here was replicated after 1000 bootstrap iterations. SaELO (Sparus aurata, AAT81404), TmELOVL5 (Thunnus maccoyii, ACZ55930), SsElovl5b (Salmo salar, NP_001130024), SsElovl5a (Salmo salar, NP_001117039), OmELO (Oncorhynchus mykiss, NP_001118108), DrELOVL5 (Danio rerio, NP_956747), HsELOVL5 (Homo sapiens, NP_068586), SsELOVL2 (Salmo salar, NP_001130025), and HsELOVL2 (Homo sapiens, NP_060240) (TIFF 58 kb)

Fig. 3

The elongation of endogenous fatty acids in pYES2 and pYES2-AsELOVL5 transformant. Here appears to be elongation of endogenous C16:1 n−7 to C18:1 n−7 irrespective of what PUFA (GLA, ARA or EPA) was fed to the yeast pYES-AsELOVL5 transformant. Endogenous FAs are also elongated in AsELOVL5 transformed cell without fatty acid supplementation (NS)(TIFF 271 kb)

Fig. S4

The confirmation for elongation of endogenous fatty acids in pYES2 and pYES2-AsELOVL5 transformant. Panel a and b show the elongated fatty acids, C20:1 n−9 (marked by asterisks) and C20:1 n−7 (next peak of C20:1 n−9) that would be produced from C18:1 n−9 and C18:1 n−7, respectively, when the AsELOVL5 is expressed without fatty acid supplementation (NS) and with substrate (GLA). The asterisks peaks are compared with FA standard mix (Sigma-Aldrich, Supelco Cat. 47015-U PUFA No.2) (TIFF 1117 kb)

Fig. S4

Panel b (TIFF 1219 kb)

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Kim, S.H., Kim, J.B., Jang, Y.S. et al. Isolation and functional characterization of polyunsaturated fatty acid elongase (AsELOVL5) gene from black seabream (Acanthopagrus schlegelii). Biotechnol Lett 34, 261–268 (2012). https://doi.org/10.1007/s10529-011-0746-x

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  • DOI: https://doi.org/10.1007/s10529-011-0746-x

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