Identification and functional characterisation of genes encoding the omega-3 polyunsaturated fatty acid biosynthetic pathway from the coccolithophore Emiliania huxleyi Olga Sayanova a , Richard P. Haslam a , Monica Venegas Calerón a,c , Noemi Ruiz López a , Charlotte Worthy a,b , Paul Rooks b , Michael J. Allen b , Johnathan A. Napier a,⇑ a Department of Biological Chemistry, Rothamsted Research, Harpenden, Herts AL5 2JQ, UK b Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth PL1 3DH, UK c Instituto de la Grasa (CSIC), Av. Padre García Tejero 4, 41012 Sevilla, Spain article info Article history: Received 1 November 2010 Received in revised form 11 January 2011 Keywords: Coccolithophore Desaturases Emiliania huxleyi Omega-3 long chain polyunsaturated fatty acids abstract The Prymnesiophyceae coccolithophore Emiliania huxleyi is one of the most abundant alga in our oceans and therefore plays a central role in marine foodwebs. E. huxleyi is notable for the synthesis and accumu- lation of the omega-3 long chain polyunsaturated fatty acid docosahexaenoic acid (DHA; 22:6D 4,7,10,13,16,19 , n À 3) which is accumulated in fish oils and known to have health-beneficial properties to humans, preventing cardiovascular disease and related pathologies. Here we describe the identifica- tion and functional characterisation of the five E. huxleyi genes which direct the synthesis of docosahex- aenoic acid in this alga. Surprisingly, E. huxleyi does not use the conventional D6-pathway, instead using the alternative D8-desaturation route which has previously only been observed in a few unrelated micro- organisms. Given that E. huxleyi accumulates significant levels of the D6-desaturated fatty acid stearidon- ic acid (18:4D 6,9,12,15 , n À 3), we infer that the biosynthesis of DHA is likely to be metabolically compartmentalised from the synthesis of stearidonic acid. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction The Prymnesiophyceae phytoplankton Emiliania huxleyi is a member of the coccolithophore family, most notable for their or- nate calcium carbonate extracellular platelets known as coccoliths. E. huxleyi is a cosmopolitan oceanic species and is almost certainly one of the most abundant organisms present in our seas (Iglesias- Rodriguez et al., 2008). E. huxleyi also forms blooms, the largest of which occur in the North Atlantic; remarkably, the refractive prop- erties of the coccoliths allows these blooms to be visualised remo- tely from orbiting satellites (Smyth et al., 2002). E. huxleyi is known to synthesise and accumulate omega-3 long chain polyunsaturated fatty acids (LC-PUFAs) such as eicosapentaenoic acid (EPA; 20:5D 5,8,11,14,17 , n À 3) and docosahexaenoic acid (DHA; 22:6D 4,7,10,13,16,19 , n À 3; abbreviated to DHA) (Bell and Pond, 1996) which are health-beneficial compounds found in marine foodwebs (Williams and Burdge, 2006). There is now good evi- dence of the importance of EPA and DHA in the human diet, with both these fatty acids playing a role in preventing cardiovascular disease and associated precursor states such as metabolic syndrome (Riediger et al., 2009). Given its global abundance, it is likely that E. huxleyi is a significant source of omega-3 LC-PUFA bio- synthesis in the marine environment. In view of the utility and value of omega-3 LC-PUFAs, there is considerable interest in understanding the biosynthesis of these fatty acids (Sayanova and Napier, 2004; Domergue et al., 2005a,b). Work over the last decade by ourselves and others have identified two archetypal enzyme activities which underpin the aerobic synthesis of EPA and DHA, namely the so-called ‘‘front- end’’ cytochrome b5-fusion desaturases (which insert double bonds between the carboxyl ground and pre-existing unsatura- tions) and polyunsaturated fatty acid-specific elongases (which condense acyl-substrates with malonyl-CoA to chain-elongate the fatty acid by two carbons) (reviewed in Venegas-Calerón et al., 2010). Examples of desaturases and elongases involved in the syn- thesis of omega-3 LC-PUFAs have been cloned and functionally characterised from a range of organisms including diatoms, oomy- cetes, fungi, mosses and animals (Domergue et al., 2002, 2003; Pereira et al., 2004; Tonon et al., 2005). The predominant sequence of enzymatic reactions required to convert C 18 fatty acids to C 20 + PUFAs commences with the introduction of a double bond at the D6-position, followed by C2-chain elongation and a second desaturation at the D5 position in the C 20 acyl chain, generating EPA from a-linolenic acid (ALA; 18:3D 9,12,15 , n À 3) and 0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.01.022 ⇑ Corresponding author. Tel.: +44 1582 763133. E-mail address: johnathan.napier@bbsrc.ac.uk (J.A. Napier). Phytochemistry 72 (2011) 594–600 Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem