1 3 Novel elongase of Pythium sp. with high specificity on D 6 -18C 4 desaturated fatty acids 5 6 7 Sukanya Jeennor a Q1 , Pattsarun Cheawchanlertfa a , Sarinya Suttiwattanakul a , Sarocha Panchanawaporn b , 8 Chanikul Chutrakul b , Kobkul Laoteng a,⇑ 9 a Fermentation Technology and Biochemical Engineering Laboratory, Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology, National Science 10 and Technology Development Agency, Thailand Science Park, Pathum Thani 12120, Thailand Q2 11 b Bioassay Laboratory, Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology, Thailand Science Park, Pathum Thani 12120, Thailand 12 13 15 article info 16 Article history: 17 Received 26 May 2014 18 Available online xxxx 19 Keywords: 20 D 6 -Fatty acid elongase 21 Polyunsaturated fatty acid 22 Substrate specificity and preference 23 Pythium sp. 24 25 abstract 26 We identified a novel elongase gene from a selected strain of the Oomycete, Pythium sp. BCC53698. Using 27 a PCR approach, the cloned gene (PyElo) possessed an open reading frame (ORF) of 834 bp encoding 277 28 amino acid residues. A similarity search showed that it had homology with the PUFA elongases of several 29 organisms. In addition, the signature characteristics, including four conserved motifs, a histidine-rich cat- 30 alytic motif and membrane-associated feature were present in the Pythium gene. Heterologous expres- 31 sion in Saccharomyces cerevisiae showed that it was specific for fatty acid substrates, having a double 32 bond at D 6 -position, which included c-linolenic acid (GLA) and stearidonic acid (STA), and preferentially 33 elongated the n3-18C PUFA. This is a report of elongase in Oomycete fungi, which displays very high spec- 34 ificity on D 6 -18C desaturated fatty acids. This will be a powerful tool to engineer PUFA biosynthesis in 35 organisms of interest through the n-6 series pathway for producing value-added fatty acids. 36 Ó 2014 Published by Elsevier Inc. 37 38 39 40 1. Introduction 41 Long chain-polyunsaturated fatty acids (LC-PUFAs) have been 42 recognized as nutritionally important lipids with vast applications 43 in the food and feed industries. In particular, the importance of 44 20-carbon PUFAs as principle precursors of biologically active 45 molecules, called eicosanoids, has been of considerable interest. 46 The series-1 prostaglandins (PGE 1 ) are derived from dihomo- 47 gamma linolenic acid (DGLA, C20:3D 8,11,14 ), whereas arachidonic 48 acid (ARA, C20:4D 5,8,11,14 ) and eicosapentaenoic acid (EPA, 49 C20:5D 5,8,11,14,17 ) are precursors for synthesis of PGE 2 and PGE 3 , 50 respectively [1]. Indeed, the 20-carbon PUFAs are categorized into 51 lipid-based biofunctional product, which display a wide range of 52 beneficial effects in eukaryotic systems, such as the cardiovascular, 53 immune and reproduction systems. In general, LC-PUFAs are syn- 54 thesized via a series of oxygen-dependent desaturation and elon- 55 gation reactions. Similar to the fatty acid desaturation systems, 56 there are multiple membrane-bound elongation systems, which 57 are responsible for chain extension of fatty acids. Differentiation 58 of elongase activities relies on their specificity towards fatty acyl 59 substrates [2]. Most PUFA-elongases have broad specificities on 60 various fatty acid substrates, containing different double bond 61 positions and acyl chain lengths. With regard to the biosynthetic 62 pathway of 20-carbon PUFAs, the elongase enzymes with specific- 63 ity for 18-carbon PUFAs are of particular interest. It has been 64 shown that the D 6 -18C-elongase is the rate-limiting step in 20-car- 65 bon PUFAs biosynthesis [3]. The selection of the elongase enzyme, 66 that is highly specific for a target substrate, is a promising strategy 67 for reducing the bypass of metabolic flux in the biosynthetic 68 pathway. 69 The Oomycete fungi, such as Pythium sp. and Phytophthora sp., 70 are able to synthesize 20-carbon PUFAs. However, there is a varia- 71 tion in their fatty acid profiles and contents that is strain-depen- 72 dent. Previously, we analyzed fatty acid profiles of a number of 73 selected strains of Oomycete fungi (unpublished data). Of them, 74 Pythium sp. BCC 53698 contained DGLA, ARA and EPA at high 75 amounts, whereas other D 6 -elongated PUFAs including eicosadie- 76 noic acid (EDA, C20:2D 11,14 ) were undetectable, presuming that 77 it might carry an elongase gene with high specificity for GLA 78 (C18:3D 6,9,12 ). As a consequence, a PUFA-elongase gene was iden- 79 tified from Pythium sp. BCC 53698 in this work. Its functional char- 80 acteristics in terms of substrate specificity and preference were 81 investigated by heterologous expression in Saccharomyces cerevisi- 82 ae. This is a report describing the identification of a novel elongase http://dx.doi.org/10.1016/j.bbrc.2014.06.004 0006-291X/Ó 2014 Published by Elsevier Inc. ⇑ Corresponding author. Address: National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand. Fax: +66 3 5646707. E-mail addresses: kobkul@biotec.or.th, laotengk@yahoo.com (K. Laoteng). Biochemical and Biophysical Research Communications xxx (2014) xxx–xxx Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc YBBRC 32244 No. of Pages 6, Model 5G 13 June 2014 Please cite this article in press as: S. Jeennor et al., Novel elongase of Pythium sp. with high specificity on D 6 -18C desaturated fatty acids, Biochem. Biophys. Res. Commun. (2014), http://dx.doi.org/10.1016/j.bbrc.2014.06.004