Biochem. J. (2004) 384
(357–366) (Printed in Great Britain)
Identification of two novel microalgal enzymes involved in the conversion of the w3-fatty acid, eicosapentaenoic acid, into docosahexaenoic acid
Suzette L. PEREIRA1, Amanda E. LEONARD, Yung-Sheng HUANG, Lu-Te CHUANG and Pradip MUKERJI
Department of Strategic Research, Ross Products Division-Abbott Laboratories, 3300 Stelzer Road, Columbus, OH 43219, U.S.A.
Marine microalgae such as Pavlova and Isochrysis produce abundant amounts of the w3-PUFAs (polyunsaturated fatty acids), EPA (eicosapentaenoic acid, 20:5n–3) and DHA (docosahexaenoic acid, 22:6n–3). The pathway leading to the conversion of EPA into DHA in these lower eukaryotes is not well established although it is predicted to involve an elongation step, catalysed by an elongating enzyme complex, leading to the conversion of EPA into w3-DPA (w–3-docosapentaenoic acid, 22:5n–3); followed by a desaturation step, catalysed by a D4-desaturase, which results in the conversion of DPA into DHA. To date, the enzymes involved in the elongation of EPA have not been identified from any lower eukaryote. In the present study, we describe the identification of microalgal genes involved in the two-step conversion of EPA into DHA. By expressed sequence tag analysis, a gene (pavELO) encoding a novel elongase was identified from Pavlova, which catalysed the conversion of EPA into w3-DPA in yeast. Unlike any previously identified elongase from higher or lower eukaryotes, this enzyme displayed unique substrate specificity for both n–6 and n–3 C20-PUFA substrates, with no activity towards any C18- or C22-PUFA substrates. In addition, a novel D4-desaturase gene (IgD4) was isolated from Isochrysis, which was capable of converting w3-DPA into DHA, as well as adrenic acid (22:4n–6) into w6-DPA. Yeast co-expression studies, with pavELO and IgD4, revealed that these genes were capable of functioning together to carry out the two-step conversion of EPA into DHA.
Key words: docosahexaenoic acid, eicosapentaenoic acid, Isochrysis, microalgae, Pavlova, polyunsaturated fatty acid.
Abbreviations used: ADA, adrenic acid; ARA, arachidonic acid; DHA, docosahexaenoic acid; DOB, dropout broth; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; EST, expressed sequence tag; FAME, fatty acid methyl esters; PKS, polyketide synthase; PUFA, polyunsaturated fatty acid; RACE, rapid amplification of cDNA ends.
1To whom correspondence should be addressed (email suzette.pereira@abbott.com).
The nucleotide sequence data reported have been submitted to DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession numbers AY630573 and AY630574.
Received 8 June 2004/15 July 2004; accepted 13 August 2004
Published as BJ Immediate Publication 13 August 2004, DOI 10.1042/BJ20040970
The Biochemical Society, London ©2004
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